Method of treating HIV infection and related secondary infections thereof

ABSTRACT

Defibrotide including its nucleic acid components and the variants thereof can be used to treat various disease conditions. Such therapeutic compounds can also be administered in combination with other nucleic acids and peptides.

[0001] This application is a continuation-in-part of application Ser.No. 08/185,416, filed Jan. 24, 1994, which is a continuation-in-part ofapplication Ser. No. 08/002,395, filed Jan. 13, 1993, which is acontinuation-in-part of application Ser. No. 07/748,277, filed Aug. 21,1991, and application Ser. No. 07/830,886, filed Feb. 4, 1992 which is acontinuation-in-part of application Ser. No. 07/815,130, filed Dec. 27,1991.

FIELD OF INVENTION

[0002] The present invention relates to a method of administering 1) thenucleic acid components identified in defibrotide or the variantsthereof 2) the nucleic acid components identified in defibrotide or thevariants thereof in combination with sequence specific oligonucleotides,3) the nucleic acid components identified in defibrotide or the variantsthereof in combination with amino acids or other protein factors, 4)oligonucleotides containing homologous sequences of HIV and cellularregulatory factors or the variants thereof, 5) the nucleic acidcomponents identified in defibrotide or the variants thereof incombination with 4), or 6) sequence non-specific oligonucleotide totreat various disease conditions including HIV infection and its relateddiseases. The present invention discloses oligonucleotides and vectorswhich can be used as therapeutic compounds according to the invention.The present invention also relates to a treatment of drug resistance.

BACKGROUND OF THE INVENTION

[0003] Defibrotide is a polyanion salt of a deoxyribonucleic acidobtained from mammalian tissue. Defibrotide is a single-strandedpolydeoxyribonucleotide with molecular weight of approximately 20 kDa(low molecular weight form) which may be obtained from bovine lung DNAby controlled hydrolysis. Patents related to its manufacture includeU.S. Pat. No. 3,770,720 directed to a process for extracting DNA frommammalian tissue, and U.S. Pat. No. 3,899,481 directed to a process forthe controlled partial degradation of DNA extracted from animal organs.

[0004] Experimental studies have been performed to investigate theactive component of defibrotide. U.S. Pat. No. 3,770,720 discloses thatthe components of defibrotide include phosphorus 8.5%, Na 9.0%, N 14.0%,deoxyribose 23.2%, total bases 34.0%, guanine 9.4%, thymine 9.4%,adenine 9.2%, cytosine 6.0%, uracil absent, Iodine, and Zinc.

[0005] Bracht et al., (Biochem. and Biophys. Res. Com., vol. 200, No.2,1994, pp.933-937) have disclosed four aptamer sequences derived from theunfractionated defibrotide DNA precursor molecule. Two aptamers(5′-GGTTGGATTGGTTGG-3′ and 5′-GGTTGGATCGGTTGG-3′) were identified bythrombin chromatography. Another aptamer (5′-GGATGGATCGGTTGG-3′) wasfound in the PCR product from the double-stranded DNA precursor. Thesequence of such aptamer was used to search the EMBL data base and wasfound in the bovine genome and Angiotensin II-AT1 receptor. The threeaptamers were found to have inhibitory activities of thrombin inducedplatelet aggregation, thromboxane biosynthesis, increase in cytosolicCa++, and fibrin clot formation. In addition, there is a non-functionaptamer (5′GGTGGTGGTTGTGGT3′) which did not display any of theactivities characteristic of defibrotide.

[0006] HIV infection is characterized by a progressive decline in immunesystem function, suppressing the infected host's ability to overcomeother, secondary infection. No cure has been found for HIV infection.The pathogenetic process in HIV infection is never unidimensional but,rather, extremely complex and multifactorial. The pathogenic progressionmay be only tangentially related to the direct infection of a giventarget cell. Death is almost inevitable, usually from an overwhelmingsecondary infection and/or HIV related neoplasm.

[0007] Current treatments for HIV infection attempt to retard theprogress of the disease or relieve its symptoms. Treatment in use todayinclude certain dideoxynucleotides such as azidothymidine (AZT orzidovudine, Burroughs Wellcome), dideoxyinosine (ddI, Bristol-MyersSquibb) or dideoxycytidine (ddC, Hoffman-LaRoche). These agents can betoxic. Their applicability is limited because of the appearance in somepatients of onerous, and sometimes lethal, side effects. These sideeffects include myelosuppression, peripheral neuropathy, andpancreatitis. In some patients, AZT has lost its effectiveness afterprolonged use. While other drugs have been proposed for treatment of HIVinfection, including the recent introduction of several HIV proteaseinhibitors, none have yet been demonstrated to be completely effective.Therefore, there remains a need in the art to develop additionaltherapeutic agents to treat HIV infection. In particular, there is aneed in the art to further identify the active components of defibrotideand their applications in various disease conditions.

SUMMARY OF THE INVENTION

[0008] It is an object of the invention to provide a method useful intreating a disease condition in a patient, such as infectious diseases,genetic diseases, degenerative diseases, DNA damage, neoplasia, and skindiseases.

[0009] To accomplish this objective, the invention provides a method oftreatment comprising administering to a patient an effective amount of atherapeutic compound comprising a nucleic acid component of defibrotide,but not including defibrotide.

[0010] Preferably, the method is practiced in a marker dependent manner,which method of treating a disease condition comprises:

[0011] (a) determining the initial state of a set of disease markers,the disease markers being observable characteristics of a patient whichdeviate from the normal condition due to the disease state and whereineach disease marker in the set has a predetermined reference range whichis indicative of the normal condition,

[0012] (b) administering to the patient a dose of a therapeutic compoundcomprising a nucleic acid component of defibrotide, but not includingdefibrotide,

[0013] (c) screening a panel of second messengers and signal transducersand selecting a repair marker, the intensity of which increasesfollowing administration of the therapeutic compound, where intensity isthe extent to which the state of the repair marker differs from itsstate in the normal condition, the repair marker being the concentrationof a compound which participates in a cellular regulatory pathway whichoperates through protein kinase A, protein kinase C, or G-protein,

[0014] (d) administering the therapeutic compound at a dose levelincrementally higher than the previous dose,

[0015] (e) repeating step (d) each time the intensity of the repairmarker increases following an incrementally higher dose,

[0016] (f) repeating steps (d) and (e) until the intensity of the repairmarker in step (c) no longer increases,

[0017] (g) administering the therapeutic compound at the highest doselevel attained in step (f) until the intensity of the repair markerreturns to the normal condition, and

[0018] (h) administering the therapeutic compound at a dose levelincrementally higher than the previous dose and repeating steps (c),(d), (e), (f) and (g) with one or more additional repair markers untilall disease markers of the set of disease markers no longer deviate fromthe normal condition.

[0019] The patient is monitored weekly for three or more weeks. Ifrelapse occurs, as indicated by a deviation of one or more diseaseand/or repair markers from the normal level, therapy is reinitiated atthe highest dose level of the prior course of therapy untilnormalization is again reached.

[0020] In a particularly preferred embodiment of the invention, themethod of treating a disease condition comprises the steps of:

[0021] (a) determining the initial state of a set of disease markers,the disease markers being observable characteristics of a patient whichdeviate from the normal condition due to the disease state and whereineach disease marker in the set has a predetermined reference range whichis indicative of the normal condition,

[0022] (b) administering to the patient a dose of a therapeutic compoundcomprising a nucleic acid component of defibrotide, but not includingdefibrotide, wherein the dose of the therapeutic compound is at a levelwhich raises a universal marker to at least five times its normal level,the universal marker being a constitutively expressed molecule which istranscriptionally activated by the therapeutic compound in all diseasestatus, and

[0023] (c) continuing to administer the therapeutic compound at the doselevel of step (b) until the universal marker returns to its normallevel.

[0024] The invention also provides a method of treating a diseasecondition via administering a nucleic acid component of defibrotide witha sequence specific nucleic acids corresponding specifically to selectedparts of the viral genome or transcriptional factors.

[0025] The invention contemplates treating HIV infection in which HIV isnot expressed and wherein the concentration of at least oneimmunological molecule, such as CD4, CD25, IL-1, IL-3, IL-4, IL-6, TNFand sIL2R, is followed. The method comprises:

[0026] (a) administering to the patient an effective amount of atherapeutic compound comprising a nucleic acid component of defibrotide,but not including defibrotide, wherein the effective amount is theamount which causes a universal marker to rise at least five times itsnormal level, the universal marker being the concentration of aconstitutively expressed molecule which is transcriptionally activatedby the therapeutic compound in all disease states, and

[0027] (b) continuing to administer the effective amount of thetherapeutic compound until the universal marker returns to its normallevel.

[0028] The present invention identifies the active components ofdefibrotide and the variants thereof. The present invention alsoprovides therapeutic oligonucleotides. Such therapeutic compounds can beused to treat various disease conditions.

BRIEF DESCRIPTION OF DRAWINGS

[0029]FIG. 1 is a diagram schematically illustrating a preferredembodiment of the invention.

[0030]FIG. 2 is a graph showing normal peripheral blood cells labelledwith 0, 10, 20 and 40 μg defibrotide-biotin combination.

[0031]FIG. 3 shows the data of FIG. 18 on a linear scale.

[0032]FIG. 4 is a graph showing the lymphocyte uptake of defibrotidewithout biotin and labelled with Cy5.18.

[0033]FIG. 5 is a graph showing the monocyte uptake of defibrotidewithout biotin and labelled with Cy5.18.

[0034]FIG. 6 is a graph showing the granulocyte uptake of defibrotidewithout biotin and labelled with Cy5.18.

[0035]FIG. 7 is a graph showing the percent expression of HIV viralproteins remaining when blood lymphocytes of an HIV infected individualwere exposed to various doses of defibrotide with and without Con-Astimulation.

[0036]FIG. 8 is a graph showing the laboratory response expressed interms of mean linear fluorescence intensity of the peripheral bloodmononuclear cells of an HIV infected individual, the cells beingsubjected in vitro to varying levels of defibrotide using a cell cultureassay technique with and without Con-A stimulation.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention provides a method for the clinicalapplications of therapeutic compounds including 1) the nucleic acidcomponents identified in defibrotide or the variants thereof 2) thenucleic acid components identified in defibrotide or the variantsthereof in combination with sequence specific oligonucleotides, 3) thenucleic acid components identified in defibrotide or the variantsthereof in combination with amino acids or other protein factors, 4)oligonucleotides containing homologous sequences of HIV and cellularregulatory factors or the variants thereof, 5) the nucleic acidcomponents identified in defibrotide or the variants thereof incombination with 4), and 6) sequence non-specific oligonucleotides.

[0038] The therapeutic compounds described in the present invention canbe employed to treat various disease conditions including HIV infectionand its related diseases. Preferably, the therapeutic compoundsdescribed in the present invention are administered in a markerdependent manner. A “marker” is an observable characteristic of apatient which may be observed directly by a clinician or determined bydiagnostic procedures. The state of an individual marker is correlatablewith the status of the disease or repair processes in the patient.Dosing of the therapeutic nucleic acids according to the method of thisinvention is based on changes in the status of these markers as taughtherein.

[0039] Treatment of various disease conditions including HIV and itsrelated disease states in accordance with the preferred method of theinvention involves the administration of a therapeutic compound of thepresent invention at a daily dose level sufficient to increase theintensity, determined as concentration or clinical observation, of amarker of cellular repair processes (“repair marker”) to a plateau level(i.e., where the intensity of the marker is not changed by continuedadministration of the therapeutic compound). This daily dose level isthe “maximum efficacious dose” for the particular disease and repairmarker. Administration of the therapeutic compound is continued at thesame dose level until the repair marker stabilizes by returning to thenormal level.

[0040] If at least one disease marker remains in an abnormal state, thedaily dose level of the therapeutic compound is increased. At least oneother repair marker will increase in intensity, and the daily dose levelis increased until the intensity of the new marker reaches a plateaulevel. Administration of the therapeutic compound is continued at thisnew maximum efficacious level until the respective repair markerstabilizes at the level and proportion assessed in normal laboratorycontrols.

[0041] When all disease markers have returned to the level of the normalstate, administration of the therapeutic compound is discontinued, butthe levels of the disease and repair markers are monitored every threeweeks, for an additional 3-6 months. If the levels of all markers remainat their normal state, cure has been achieved. If any marker deviatesfrom normal at the end of any three week period, administration of thetherapeutic compound is resumed at the highest “maximum efficaciousdose” that has been used during the immediate prior treatment, and thenew maximum effective dose is established by the known methodology.

[0042] The term “maximum efficacious dose” is defined herein as thedaily dose rate, that will elicit, in nearly 100% of treated patients,the reversal of the respective disease markers to the uniformly normallevel, and establishment of normal cellular markers. The maximumefficacious dose is usually expressed as amount of therapeutic compoundadministered per kilogram body weight per day (DKGD). The maximumefficacious dose represents a novel concept of administering apharmaceutical agent in therapeutic medicine.

[0043] The term “maximum therapeutic dose” is defined herein as thetotal cumulative dose (the daily dose summed over the duration ofadministration) that will elicit in nearly 100% of treated patients theirreversible and complete normalization of the respective diseasemarkers and resumption of normal cellular functions, i.e., the state ofcure.

[0044] The term “minimum efficacious dose” is used herein to refer tothe dose used in the heretofore universal practiced method ofadministering a pharmaceutical agent. The minimum efficacious dose isthe dose (daily dose or steady state level) that will elicit aparticular pharmaceutical action in a certain percentage of patients,without inducing the pleiotropism of the whole repair process.

[0045] Pharmacological agents have heretofore been administered at setdose levels (i.e., the “minimum efficacious dose”) to treat the grosspathology and discontinued when complete or partial remission of thegross pathology was achieved. Treatment according to the preferredmethod of this invention begins at the gross pathology stage which hasone or more associated markers. Normalization or improvement of thosemarkers indicate that the treatment is beneficial. However, such aremission is not the event which causes discontinuation of therapy inaccordance with this invention. Normalization of the markers of grosspathology indicates, rather, that a disease state corresponding to alower level of disease activity has been reached. Markers of that stage(i.e., the lower level of disease activity) are identified and treatmentis continued to normalize those markers. Complete cure is reached onlyif all stages of the revival process are treated.

[0046] The term “maximum tolerable dose,” as used herein, is defined asthe highest daily dose that can be administered without anycomplications, e.g., no bleeding complications or thrombopathy, etc.This in fact has been the sole and primary side effect of thehigh-molecular weight nucleic acid (defibrotide) utilized in the studiesreported herein, i.e., the antithrombotic effect inducing bleedingcomplications at 300 mg/kg/day dose or above. If the maximum efficaciousdose should be higher than the maximum tolerable dose, chemicalmodification of the nucleotide for more efficacious transmembranetransport and cellular entry would be necessary.

[0047] It has been determined that the therapeutic compound will notindefinitely increase transcriptional activity with increasing doses. Inthis regard, transcriptional activity will shut off when the repairmolecules are no longer needed, i.e., when no more “injury signal” istransmitted via stimulation of adenylate cyclase, second messengers,etc. In contrast, no matter how high the dose range in the normalindividual may be, there is no induction of transcriptional activity (asindicated by, e.g., elevation in the von Willebrandt antigen (vWAg)levels). This supports the fact that no complications are seen withtherapy using nucleotides which modulate cellular repair mechanisms fora therapeutic effect. For example, tissue plasminogen activator antigen(AgTPA) will not continue to rise indefinitely with increasing doses butwill increase only in the presence of injury and at the locality of theinjury, e.g., the existence of a thrombus which inevitably will beassociated with the endothelial cell of the locality specific lesion.Hence no bleeding complications are to be seen secondary to systemicinduction of the therapeutic compound at physiological dose rangesbeyond the upper limits of the prior art thrombolytic therapy.

[0048] This mechanism is supported by the way the cell modifiesactivation of the repair process. As is well known, 50% occupation ofcell surface receptors will lead to 50% increase in the baseline levelof intracellular cAMP, 100% occupation of cell surface receptors willlead to a 100% increase in the intracellular cAMP level. This willcorrespond to 5 times the elevation of the baseline vWAg level.Phosphorylation of various different transcriptional factorssimultaneously will lead to concurrent tissue specific turning on or offof the respective transcriptional factors, e.g., some molecules areturned on and some are turned off. This constitutes the pleiotropism ofthe nucleic acids as herein defined.

[0049] Treatable Disease States

[0050] Various disease conditions including HIV infection and itsrelated diseases characterized by injury-based alteration in theproduction, expression or activity of compounds whose production,expression or activity is regulated by the cell at least in partthrough 1) cell surface receptors such as Adenosine A, and A₂, collagen,thrombin, epinephrin and norepinephrine receptors, 2) through theprotein kinase A, protein kinase C, phosphorylation, or receptortyrosine kinase pathway, 3) through cytokine-receptor superfamily andregulatory factors encoded by oncogenes, or by 4) protein factors whosephosphorylation affects genomic translation and transcription may betreated with the therapeutic compounds of the present invention in amarker dependent manner as described herein.

[0051] Treatable disease states include 1) infectious diseases such asHIV infection, Protozoa infection, Schistosima infection, SchistocercaLeishmania infection, e.g., Schistosoma japonicum infection, Trypanazomainfection, e.g., Trypanozoma Cruzi infection, fungus infection, e.g.,Candida tropicalis and Candida Albicans, Aspergillus infection,Pneumocystis carinii infection, Malaria, Plasmodium vivax, gram negativebacterial infection, Cytomegalovirus infection, Hepatitis virusinfection, human papilloma virus infection; 2) genetic diseases such asDuchenne's Muscular Dystrophy, Down's Syndrome; 3) degenerative diseasessuch as encephalopathy, dementia, Alzheimer's disease, Parkinson'sdisease, neuropathy, cardiomyopathy, aging, Kearn's Sayre syndrome,retinitis pigmentosa, ataxia, seizures, proximal muscle weakness,leber's hereditary optic neuropathy, optic neuritis, radiation damage;4) neoplasia such as lympho-proliferative diseases, lymphomas, Kaposi'ssarcoma, pancreaotic cancer, neuroblastoma, leukemia, bladder carcinoma,breast cancer, skin cancer, lung cancer, colon cancer, and 5) skindiseases such as molluscum contagiosum, bacillary angiomatosis,seborrheic dermatitis, psoriasis, Reiter's syndrome, insect bitereactions, Staphylococcal folliculitis, Eosinophilic folliculitis.

[0052] The methodology described herein has universal application withinthe scope of disease states characterized by the absence or inadequacyof one or more of those cell functions which are normally regulatedthrough the cellular mechanisms listed above so long as theabnormalities in these cell functions are yet still reversible. Themethodology is also applicable to the disease states characterized byacquired or genetic dismodulation, and/or transformation. Revival,institution or reinstitution of the normal state of those functions is,by definition, a state of cure. Revival of the normal cell functions canoccur where the diseased cell preserves the biological capacity for thephysiologically predefined events of the cellular repair functions ofthe recovery process, if those events are pharmacologically induced bythe correct use of the therapeutic nucleic acids. Complete cure is thetherapeutic objective. The decisive factor in the success of thistherapeutic approach is not only the pharmaceutical agent, but how it isutilized. If the biological capacity for regaining normalcy is there,therapeutic failure is eliminated. This biologically predeterminedpotential for cure is reproducibly and predictably obtainable, however,only by the correctly determined iatrogenetically controlled doselevels, and duration of therapy. Incorrect dose administration leads tothe therapeutically missed event of complete cure. Complete cure,however, is not possible if necessary dose levels cannot be attainedwithout complications such as bleeding or thrombopathy.

[0053] While the marker dependent dose methodology is universallyapplicable, it has been surprisingly discovered that HIV, as well asassociated opportunistic secondary infections can be effectively treatedwith the therapeutic compounds described in the present invention.

[0054] Therapeutic Compounds

[0055] The therapeutic compounds contemplated in the present inventioninclude 1) sequence non-specific oligonucleotide, 2) nucleic acidcomponents of defibrotide, 3) variants and derivatives of 2), 4)sequence specific nucleic acid in combination with 2), 5) amino acids orprotein factors in combination with 2), 6) oligonucleotides containinghomologous sequences of HIV sequence and other genes encoding cellularregulatory factors.

[0056] Sequence non-specific oligonucleotides of the present inventionis an oligomer or a polymer of deoxyribonucleotides or derivativesthereof. The compound may be native or chemically synthesized, or afragment of a native polydeoxyribonucleotide. The compound has at leastthree nucleotide residues, and may have up to about 250 residues.Preferably, the nucleotide compound will have from about 15 to about 200residues, more preferably from about 20 to about 150 residues, mostpreferably from about 50 to about 75 residues. The sequence of thenucleotide residues in the polymer is not critical, and may includeinterdisposed sense, anti-sense, non-sense or missense sequences. Atherapeutic composition may contain polynucleotide molecules withvarying numbers of residues within the range described above. Theskilled worker will be able to select an appropriate length (degree ofpolymerization) based on the ability of the compound to penetrate thecell and on the ability of the compound to cause a change in the levelof various repair markers in accordance with the method of thisinvention.

[0057] The nucleic acid compound will preferably be relatively resistantto ecto- and endonucleases. The 3′ OH of the terminal residue of thetherapeutic compound according to this invention may be phosphorylatedor not, and the compound will still function without the need forintracellular phosphorylation. The therapeutic compound according tothis invention is a polyanion, and the negative charge is balanced bycounter ions. The counter ions may be alkali metal ions or alkalineearth ions, biologic amines or other suitable counter ions which do notinterfere with treatment according to the method of this invention.Preferably, at least some of the counter ions are zinc ions. The amountof zinc, however, may be increased either be directly incorporating zincinto the nucleotide compound or, alternatively, by administering zinc,e.g., in the form of a dietary supplement, along with the therapeuticnucleotide. Zinc containing compounds may be coadministered with thenucleotide to obtain a ratio of from 2-20 zinc atoms per phosphate groupor iodine atom.

[0058] Defibrotide may be obtained from mammalian tissues as describedin U.S. Pat. No. 3,770,720 or obtained from commercial source, e.g.,CRINOS Farmacobiologica S.p.A., Villa Guardia (Como), Italy. Any meansknown in the art may be used to analyze the nucleic acid components ofdefibrotide. Usually, HPLC can be used to separate defibrotide into itsnucleotide and oligonucleotide components. For instance, inreversed-phase HPLC, defibrotide may be run on a Vydac C8 or C18analytical HPLC column using a Rainin HPLC system. The flow rate couldbe set at 1 ml/min and the eluent can be monitored at 260 nm and 280 nmwavelengths. Such column run may be carried out isocratically using 0.1TFA in water. In some runs, peaks can be collected for subsequent massspectrometry.

[0059] It is a discovery of the present invention that all defibrotidecomponents are eluted in approximately 8-10 peaks within 10 minutes. Inorder to identify the nucleotide composition of defibrotide, the mono-,di-, tri- and cyclic monophosphates of T, C, G, A, and U may bechromatographed under conditions identical to those used fordefibrotide. If the retention time for a purified nucleotide issuperimposeable (±0.1 min) on a defibrotide peak, it can be taken asevidence for the putative presence of such nucleotide in defibrotide.Peaks collected from HPLC runs may be concentrated by vacuum evaporationand be analyzed in mass spectrometry. All mass spectra may be collectedon a matrix assisted laser desorption ionization-time of flight(MALDI-TOF) Voyager Biospectrometry Workstation (Perseptive BioSystems)and run in the negative ion mode.

[0060] It is a discovery of the present invention that a simple HPLC C8column elutes with 0.1% TFA in water provides the best separation ofdefibrotide obtained through commercial source, i.e., Noravid (CRINOSFarmacobiologica S.p.A., Villa Guardia, Italy). Defibrotide elutes froma C8 column in approximately 10 peaks with retention times betweenapproximately 3 and 9 minutes. One of the peaks, i.e., peak number 4represents two 25-30 mer oligonucleotide with molecular weight of about8171.58 and 8433.75 Dalton, respectively. Only routine experimentationsare needed to sequence the two 25-30 mers.

[0061] The nucleic acid components of defibrotide include allnucleotides and/or oligonucleotides identified in defibrotide whichinclude but are not limited to dCTP, dATP, dGTP, dTTP, dAMP, dGMP, dCDP,dADP, ATP, AMP, CTP, CMP, UTP, cyclic TMP, cyclic UMP, cyclic GMP,oligonucleotides containing from 6 nucleotides to less than 60nucleotides, aptamer #1 GGTTGGATTGGTTGG (SEQ ID NO:1), aptamer #2GGTTGGATCGGTTGG (SEQ ID NO:2), aptamer #3 GGATGGATCGGTTGG (SEQ ID NO:3),and aptamer #4 GGTGGTGGTTGTGGT (SEQ ID NO:4), and two 25-30 meroligonucleotides with molecular weight of about 8171.58 and 8433.75dalton respectively and identified via HPLC analysis as discussed above.

[0062] Any variant of the nucleic acid components can also be used asthe therapeutic compounds in the present invention. Variants includeoligonucleotides having complete or partial sequence homology with theoligonucleotides of defibrotide. Variants include nucleic acid fragmentcomprising the oligonucleotide sequences identified in defibrotide. Forexample any DNA fragment containing the oligonucleotide sequence ofdefibrotide and additional sequences at the ends of the oligonucleotidesequence is contemplated in the present invention as a variant. Normallythe number of additional nucleotides at the ends is from 1 to 100,preferably from about 5 to 50, more preferaby from about 10 to 30.

[0063] The homology level may be at least from about 50% to about 70%,preferably 80% to 90%, more preferably 95%. The homologous region may becontinuous or scattered through out a nucleotide fragment. For example,aptamer #1 of defibrotide (5′-GGTTGGATTGGTTGG-3′) has complete andpartial homology to several genomes, e.g., Schizosaccharomyces, pombeGATA-binding region, and Streptococcus pneumonia Dpn I gene. Aptamer #2of defibrotide (5′GGTTGGATCGGTTGG-3′) has homology to several genomes,e.g., Mycobacterium leprae cosmid B0462.

[0064] Aptamer #4 of defibrotide (5′-GGTGGTGGTTGTGGT-3′) has homology tovarious genomes, e.g., chicken liver cell adhesion molecule, humangelanin receptor mRNA, Schistosoma japonicum eggshell protein,Schistosoma japonicum ESG-1 protein mRNA, human m RNA with TGG repeatclone 83, Schistosoma japonicum ESG-2AA protein mRNA, Candida tropicalisPOX9 gene, Candida Tropicalis cat gene, Schistocerca americanaAntennapedia, chicken liver cell adhesion molecule, human papillomavirus type 20, homo sapiens mitochondrial genome, gorilla mtDNA, humanmtDNA, human DNA sequence from cosmid U157D, Leishmania major cosmidclone L2759, Plasmodiun vivax Serine repeat antigen, P. clarkii mRNA,Trypanosoma cruzi mucin-like protein, L. major mRNA for surface antigenP2, Aspergillus aculeatus (clone PC1G1), Candida Albicans DNA for MNT2gene, E. Coli K-12 genome, Mouse amyloid beta precursor, CandidaAlbicans topoisomerase type, human homolog of Drosophila spilicing gene,E. Coli gcvh gene 3′end, human Down Syndrome region of Chromosome, E.Coli gcv operon gene sequence, Drosophila melanogaster receptor proteinand polyheamotic DNA, human Papilloma virus type 25 genomic, Drosophilamelanogaster Zn finger, Pneumocystis carinii, Dystrophin associatedprotein of Duchenne's muscular dystrophy, (Sequence 7 from U.S. Pat. No.5,449,616), and DNA Polymerase (Sequence 14 from U.S. Pat. No.5,556,772).

[0065] Variants of aptamer # 4 also include homologous sequences of HIVand aptamer #4. For example, homologous sequences may be found ingag/pol, c-vif, or env regions of HIV. Particular homologous sequencesmay be found on three sites on gag/pol HIV genome region. Thetranslation of the aptamer #4 region on gag site is a peptide “PEPTA”,and the pol gene fragment translates the same DNA sequence into ′TRANS.In a preferred embodiment, S-Oligo variants of aptamer 1)5′GGGCTGTTGGCTCTGGTCTGCTCTGAAGGAAATTCCCTGGCCTTCCCTTG3′, 2)5′ACCAGAGCCAACAGC3′, 3) 5′CCTGGCCTTCCCTTG3′.

[0066] Variants of aptamer #4 also include homologous sequences of agene encoding a cellular regulatory factor and aptamer #4. Examples ofsuch homologous sequences are listed in Table 1. TABLE 1 Examples ofSequences Homologous to Aptamer #4 Cellular Factor Strand HomologyRegion Match Percent human myc anti-S 2345-2449 13 86.6 TNF-Receptoranti-S 5039-5053 12 80.0 anti-S  036-950 12 80.0 anti-S 5574-5588 1173.0 anti-S 5045-5059 11 73.0 S 2577-2591 11 73.0 human TNF S  693-70711 73.0 S 1149-1163 11 73.0 S 1034-1048 11 73.0 bTNFg S 6375-6389 1066.6 S 5723-5737 10 66.6 S 4195-4209 10 66.6 S 4186-4200 10 66.6 S1878-1892 10 66.6 S 1875-1889 10 66.6 S 1872-1886 10 66.6 S 1591-1605 1066.0 S 1565-1579 10 66.0 bTNFfg anti-S 5638-5652; 5355-5369; 10 66.65046-5060; 4447-4461; 4435-4449; 4224-4238; 4221-4235; 1990-2004;1125-1139; 933-947;  930-944; 119-133. hsch-ras anti-S  15-29 12 80.0 18-32 bt-ras S 1404-1418 10 73.0 1401-1415 anti-S  978-992 10 66.6 975-989 hsch-ras anti-S  12-26 10 66.6  21-35 human-ras-md anti-S 28-42 10 66.6  15-29 human-abl S 3253-3267; 3064-3078; 10 66.63061-3075; 3058-3072; 2918-2932; 2195-2209; 2189-2203; 1952-1966;1059-1073. human bcl S 3253-3267; 3064-3078; 10 66.6 3061-3075;3058-3072; 2918-2932; 2195-2209; 2189-2203; 1952-1966; 1050-1064. anti-S3451-3465; 1366-1380; 1198-1212; 120-134;  270-284. human fos S5166-5180; 3358-3372 10 66.6  828-842 anti-S 6019-6033; 5561-5575;4901-4915; 3695-3709;  361-375; 98-112; 35-49. human IL-1 S  247-261 1066.6  244-258 anti-S  920-934 10 66.6  281-295 Musnos S 3717-3731;3685-3699; 10 66.6 (nitric oxide 2904-2918; 2669-2683; synthase)2408-2422; 2007-2021. anti-S 2806-2820; 2572-2586; 2530-2544; 1720-1734;1717-1731; 1237-1251; 1075-1089; 504-518.

[0067] Variants of aptamer #4 also include homologous sequences ofmitochondrial DNA and aptamer #4. Aptamer #4 has 100% homology to NADHDehydrogenase Subunit 6 at target site of 13741; homology of 86.6% totRNA glu. at target site of 14101; homology of 80% to NADH Dehydrogenasesubunit 4 at target site 10249; homology of 80% to 16 S rRNA at targetsite of 1924; homology of 73.3% to D-loop at target site of 16470;homology of 73.3% to NADH Dehydrogenase Subunit 5 at target site of14227; homology of 73.3% to NAD Hydrogenase Subunit 6 at target site of13819; homology of 73.3% to NADH Dehydrogenase of Subunit 6 at targetsite of 13744; homology of 73.3% to NADDhydrogenase Subunit 5 at targetsites of 13467 and 11763; homology of 73.3% of NAD Dehydrogenase ofSubunit 6 at target site 10246; homology of 73.3% to cytochrome oxidaseSubunit 3 at target site of 8820; homology of 73.3% to cytochromeoxidase subunit 6 at target site 8327; homology of 73.3% to ATPasesubunit 8 at target site 7810; homology of 73.3% to tRNA-lys at targetsite of 7752; homology of 73.3% to cytochrome c oxidase subunit 1attarget sites 5961, 5478; homology of 73.3% to NAD Dehydrogenase Subunit2 at target sites of 4871, 4733, 45594145; homology of 73.3% to NADHDehydrogenase Subunit 1 at target sites of 2922, 2919; and homology of73.3% to 16S rRNA at target sites of 1936, 1635.

[0068] Derivatives of the nucleic acid components are contemplated inthe present invention. Derivatives include the nucleic acid componentsconjugated with poly(L-lysine) or modified by, for example, the additionof amino acids such as lysine, histidine and arginine, the addition ofoptimum concentrations of folate and/or biotin, the addition of theoptimum ratios of metals and ions including zinc, manganese and iodine,by the addition of 5′-polyalkyl moieties, cholesterol, vitamin E,1-2-di-O-hexadecyl-3-glyceryl and other lipophilic moieties and/ormodified by the replacement of phosphodiester bonds with phosphothiotatebonds, and/or modified nucleotide sequences of the prototype nucleicacid, defibrotide.

[0069] Derivatives of the nucleic acid components of defibrotide alsoinclude modified nucleic acid components. Any modification method knownin the art may also be employed to modify the nucleic acid components ofdefibrotide. For example, addition of RNA monomer, i.e., adenosine onthe 3′ end of a DNA oligonucleotide by using an RNA-3′ solid supportwith (di)phosphorodimite chemistry; insertion of adenine,deoxyadenosine, or dA adnine base in a oligonucleotide; insertion of 5′monophosphate, e.g., 5′-P-A-C-G-T or 3′ monophosphate, e.g.,A-C-G-T-P-3′ at any selected spot on an oligo using (di)phosphoramiditechemistry; addition of any nucleotide on the end of tri-phosphates, e.g.N-P-P-P-A-C-G-T; production of di-nucleotides, e.g., N-5′-P-P-P-P-5′-N;conjugate NTP to any oligonucleotide, e.g., N-5′-P-P-P-P-5′; coupling ofcyclic nucleotides, e.g., use of APPPPA-synthase to makeA-5′-P-P-P-P-5′A; membrane support modifications including addition ofcholesterol to any position of an oligonucleotide with(di)phosphorodimite chemistry; addition of peptides via carboxy-dT toany position on an oligonucleotide (carboxy-dT can be coupled directlyto a molecule containing a primary amino group using peptide chemistryor via the intermediate N-hydroxysuccinimide (NHS) ester); attachingmolecules to any site on an oligonucleotide using amino linkers andlinker-spacers via NHS ester chemistry; linking oligonucleotidestogether, e.g., 5′-5′ or 3′-3′ with (di)phosphorodite chemistry;thiolated, methylayed, or in the form of propyne oligonucleotidesantisense oligonucleotides produced via (di)phosphorodite chemistry;multiple symmetric (same sequence) or asymmetric (different sequence)braced oligonucleotides with targeted virus and subtargeted cellularelements via (di)phosphorodite.

[0070] Oligonucleotides containing a homologous sequence of HIV and agene encoding a cellular regulatory factor are also contemplated in thepresent invention. The sequence homology between HIV and other cellularregulatory factors may be at least 40%, preferably at least from 60% to70%, and more preferably from 80% to 90%. The length of the homologyregion may be from 3 nucleotides to 100 nucleotides, preferably from 6to 60 nucleotides. The cellular regulatory factors include transcriptionfactors, oncogene products, and any factors involved in the signaltransduction pathway, e.g., TNF receptor, RIP, IL-2 receptor, IL-1analog, TNF-α, c-myc, c-abl, c-fos, c-ras, dystrophin, surfaceglycoprotein proteins of L-CAM and cathedrin, and B-myb. Table 6 lists afew examples of such oligonucleotides. TABLE 6 Examples ofOligonucleotides Oligonucleotide Homology Region in HIV CellularRegulatory Factor CAGCTGCACCTGCCAAGC gag/pol 968-984 human TNF receptorATAAAATATACCATATACA gag 2315-2331 human RIP protein kinase (HSU50062)TCATAAAATATACTATATTCA gag 2312-2331 mouse TNF receptor (mmu 25995)ATATTAAAGAACGCTGTTTACAAT vif 4847-4876 IL-2 receptor ACTTGGATGCAGTTGTGAAGAGAA env 7485-7502 TNF receptor (cell death proteinHSU25994) AATTAAGGCATAAGAAAACTAAGA env 6118-6150 IL-1 analog AATATGCACTCTCTCCCTCAAGGACTCAGCTTT 5′ untranslated region pos. TNF-α promoterCTGAAG 60-90 CAATAATAAAAGGGGAAA gag 186-203 c-myc AGTGCAACCGGCAGGAGGTGA5′ untranslated region pos. c-abl 88-109 GCCACCAGCCCCTCCCCAGACTCT pol1522-1547 B-myb, c-myc CAGGTGGAGGCAACAG protooncogene

[0071] Defibrotide or the nucleic acid components of defibrotide andvariants thereof may be administered in combination with 1) sequencespecific nucleic acids, 2) amino acids, 3) protein factors, 4) sequencespecific nucleic acids and sequence non-specific nucleic acids, 5)sequence specific nucleic acids and sequence specific peptides includingbut are not limited to peptides encoded by oligonucleotides ofdefibrotides and the variants thereof, 6) sequence specific nucleicacids and sequence non-specific peptides.

[0072] The sequence specific nucleic acids include but are not limitedto anti-protease sequences, retroviral promoter sequences, TARsequences, HIV mutants of TAR decoy RNA, mutants TAR decoy RNA, negativemutants of the viral REV transactivator, synthetic promoters with theconsensus sequence for binding of the transcription factor Sp1 and theTATA box, mutants of TATA box, TAT mutants wherein the mutationsinvolving the seven cysteine residues, sense, anti-sense, missensederivative of CIS acting negative elements (CRS) present in theintegrase gene and REV mutants, transdominant suppressors of REV(mutations involving amino acid 78 and 79), NEF-cDNA sequences and itsmutants with or without U3 region sequence of the 3′LTR, POL reversetranscriptase gene mutants, POL viral integrase gene and its mutants,POL viral protease gene mutants, HIV-I LTR enhancer (−137 to −17)mutants, HIV LTR promoters starting at −78, HIV LTR sequences encoding aarginine fork from aa27 to aa38, HIV-I LTR sense sequences of thenegative regulatory element (−340 to −185), HIV-1 LTR consensussequences for binding of transcription factors of AP1/COUP, NFAT-1, USF,TCF-α, NF-KB, TCF-1a, TBP, and inhibitors of the consensus sequence, LTRNFkB mutants (−104 to −80), LTR Sp1 (GC box) binding site and TATA boxmutants, LTR GAG gene sequence mutants, LTR mutants (454 to +180), LTRgenomic repeats at +80, LTR regions responsive for cellulartranscription factors between and to the left of U3 to 454 extending to−7, 3′ LTR and its variants, 5′ LTR and its variants, LTR variants,inhibitors of UBP-1 or LBP-1 binding sequence (−5 to +82), ENV, GAG, POLgene sequences placed 3′ of the REV mutant codon, short sequence mutants(15-60 mer), and host DNA sequences of preferred targets for proviralintegration.

[0073] Amino acids administered in combination with the nucleic acidcomponents and the variants thereof include these involved in signaltransduction pathways and phosphorylations. They include but are notlimited to threonine, serine, tyrosine, and proline.

[0074] Protein factors administered in combination with the nucleic acidcomponents and the variants thereof include DNA polymerase, proteaseinhibitor, and reverse transcriptase inhibitor.

[0075] In addition, N-containing ring compounds, e.g., pyrimidine,purine, adenylic, and guanosine can also be administered in combinationwith the nucleic acid compounds and the variants thereof in the presentinvention.

[0076] The homologous sequences of HIV and a gene encoding a cellularregulatory factor may also be administered in combination withhomologous sequences of nucleic acid components of defibrotide and thevariants thereof, especially the homologous sequences of aptamer #4 anda gene encoding a cellular regulatory factor.

[0077] The nucleic acid compounds of the present invention can beadministered directly or via a vector. Any vector capable of replicatingin vivo can be used to carry the nucleic acid compounds. Preferably, thevectors employed are suitable for gene delivery. Expression/replicationvectors are readily available in the art, e.g., pCI, pCI-neo.

[0078] It is a discovery of the present invention that mitochondrialgenome, especially origin of replication, e.g., from a human can be usedto construct replication/expression vectors. Preferably, 5′ end ofmitochondrial 12S RNA containing sequences from about nucleotide 72 to1025 and mitochondrial DNA containing sequences from nucleotide 1 to 72can be used.

[0079] In replication/expression vectors, the oligonucleotides of thepresent invention can be flanked by some buffer sequences. In apreferred embodiment, pCI-neo vector can be cut by Bgl2 and BamH1, andeIF-4E initiation factor gene may be inserted. The sequence of eIF-4Egene specifically relevent to such vector isGGCCAGGCATGGTAAGTCATACCTATAATCCCAGCACTGTGGGAGGCCAAGGAAGGGGGATCCCTTGAGCTCAAGAGTTTAAGACCGAGATCGAT (upstream of Alu) andAAAGAGTTTAAGACCAGCTTGGGCAACACAGTCAGACTTCATCTCTATAAATAATTTAAAAATTAGCCAAGCATGGTGGCGTGGTACCCTTGTGGGTTCCAGGCTTATTTGGGAGGTTGAGGTAAAGGAATTCTCTTGGACGCCCAGGTAGTCAAGGTTGCAGTGAGCCATAATCAAACCACTGCACTCCAGCATGGCAACAGAGCAAGACCCCATCTCAAATATAT (downstream of Alu).

[0080] Subsequently, the oligonucleotides of the present invention canbe inserted into the eIF-4E gene, preferably at the Alu site of eIF-4Egene.

[0081] In replication/expression vectors, the oligonucleotides of thepresent invention can be driven by a promoter, especially a TARpromoter, a HIV LTR promoter, or a promoter of DNA polymerase.Alternatively, the oligonucleotides of the present invention can beco-expressed or co-replicated with a gene encoding DNA polymerase. Tatprotein may be added to enhance vector replication.

[0082] The mitochondrial vectors discussed above can also be used tosupply oligonucleotides with wild-type mitochondrial sequences. HIVpatients are likely to have mutations in mitochondrial DNA, e.g,cytochrome-oxidase (COX) gene, NADH subunits, origin of replication,D-loop, t-RNA lysine, tRNA glu, and ATPase subunits. It is routine toscreen these genes for mutations. Upon finding of mutations inmitochondrial DNA, oligonucleotides containing the correspondingwild-type mitochondrial DNA sequences can be administered to treat thedisease conditions associated with such genetic alterations.

[0083] It is also a discovery of the present invention that a drugresistance can be treated via administering the nucleic acid componentsof defibrotide and the variants thereof in combination with the drug,e.g., a protease inhibitor.

[0084] Marker-Driven Therapy

[0085] The claimed method involves the use of a “marker dependent doseassessment” methodology for determining the therapeutically mostefficacious use of the respective pharmaceutical agents. The use ofincremental marker stratification reflects the concept that “maximumefficacious dose” is redefined through the different stages oftreatment, each time adjusted to the respective specific marker mostrepresentative of the respective pathogenic/clinical picture of thedisease state. Treatment at respectively higher doses corresponding tothe progressively lower disease activity levels are continued until astate of total cure is reached.

[0086] Intrinsic to the claimed method is the total elimination ofempirically assessed doses or constant therapy doses, arrived at by theuniversal pharmaceutical principal of “minimum efficacious dose” for aclass of drugs, which, until the present time, has been the standard forthe definition of the “effective therapeutic dose.” The respective dosesthereof are defined to elicit a response corresponding to differentdisease functions of the treated cell and revival of the respectivedisease parameters, in a stratified fashion.

[0087] The method of treating various diseases provided by thisinvention uses specific clinical and laboratory markers to assessdosages to be administered. The markers vary from gross clinicalobservations of pathology to the progressively subclinical yet validdetection of certain laboratory levels associated with a particulardisease. The preferred markers are the clinical parameters as well asthe molecular products produced, or inhibited, present or absent whencellular events associated with a particular disease occur.

[0088] Certain laboratory assays are used to assure that the dosages aresafe for the patient being treated. For therapy with defibrotide thesemay include prothrombin time, activated partial prothromboplastin time,thrombin time, reptilase time, bleeding time, platelet function assays,and coagulation factors. A second set of laboratory assays (i.e.,“disease markers”) are utilized to indicate the efficacy of the doses.“Repair markers” are used to assess clinical adequacy of dose escalationand duration of therapy.

[0089] As defined herein, “normal cellular markers” are molecules ofnormal cellular function. They are tissue and cell specific and mayshare common pathways of second messengers or signal transductionpathways and normal cellular genomes. At the genome level, normal cellmarkers are genes that are constitutively expressed, transcribed,translated and transduced. Establishing dose and duration of therapybased on second messengers, signal transduction pathways and inductionof genomic transcription is a novel modality of administering apharmaceutical agent.

[0090] As defined herein, “disease markers” are markers which areinduced and defined by the type of disease process. Disease markers areclinical or laboratory parameters that deviate from normalcy. A diseasemarker may be absent or present, decreased or increased. At the genomelevel, disease markers are genomes of genetic dismodulation (e.g. viralgenome, transcribed oncogenes, mistranscribed genomes); nontranscribedgenomes (e.g., familial/genetically absent genomes,under-regulated/suppressed genomes), and/or over-expressed, notappropriately shut off transcriptions of genomes (e.g. activated repairmolecules, second messengers and molecules of signal transductionpathways).

[0091] Disease markers are observable characteristics of the organismwhose status in a disease state differs from the status in the normal(non-disease) state. Such characteristics and their association withtheir respective disease states are well known to the skilledpractitioner. In the practice of the method of this invention, it iscontemplated that the practitioner will monitor the status of multipledisease markers related to the disease being treated, eithersimultaneously or sequentially. The disease markers include bothclinical markers, which are observed directly by clinician, andlaboratory markers, which represent quantitative values determined bysupport staff. These characteristics include, but are not limited to,the concentration of compounds whose production or expression isaffected by injury-based alteration of cell surface receptors such asAdenosine A₁ and A₂, collagen, thrombin, epinephrin and norepinephrinereceptors, of protein kinase A or protein kinase C pathways, or ofprotein factors whose phosphorylation affects genomic translation andtranscription, or hybridization of genomic enhancers/inhibitors infusionor excess enhancers, infusion of excess genomes to depleteviral/cellular transactivation transcription factors, etc. where theconcentration in the disease state differs from the concentration in thenormal state. Disease markers for HIV related disease states includeodynophagia, arthralgia, Herpes labialis, Herpes genitalis, cryptococcaldiarrhea, Karnofsky performance score, waste syndrome.

[0092] The normal state concentration of these markers will be known tothe skilled practitioner, and usually represents a range ofconcentration values determined by measurement of the concentration ofthe compound in a large number of individuals who are not in a diseasestate, by the respective laboratory.

[0093] Repair markers are compounds that participate in the regulatorypathways which include protein kinase A or protein kinase C. Adenylatecyclase is known to be activated by G-proteins (see Ross, 1992, CurrentBiology, 2(10):517-519, the disclosure of which is incorporated hereinby reference) with eventual production of cAMP and cAMP-dependentactivation of protein kinase A, leading to phosphorylation of therespective transcription factors, until 100% of the cell membranereceptors are taken up by the ligands. For defibrotide these receptorsare β-adrenergic receptors, collagen receptors, adenosine A₁/A₂receptors, ADP receptors, thrombin receptors, collagen receptors, etc).A parallel pathway operates through activation of protein kinase C, inresponse to intracellular calcium ion level, inositol triphosphate anddiacylglycerol, responsive to ligand binding to another set of receptorsand similarly controlling transcription/translation of respectiveproteins. These pathways, and their intermediate compounds are wellknown to those skilled in the art. However, their use in assessment oftherapeutic dosage have not, heretofore, been known in the art.

[0094] In particular, “repair markers” are molecules in the pathways ofthe respective cellular repair processes defined by the type of injury.Repair markers are transcribed or shut off genes, second messengersand/or molecules of the signal transduction pathways that may beincreased, decreased, or absent in response to cellular injury. Asdiscussed herein, the term “repair marker” may refer to the compound orits concentration or the measurement value of an assay associated withthe concentration of the compound. Examples of suitable repair markersinclude but are not limited to cAMP, cGMP, IL-1, IL-2, TNF-α, IL-6,cGMP/cAMP ratio, total lymphocyte count, T lymphocyte count, CD4 count,CD8 count, cAMP dependent protein kinase A enzyme, adenylate cyclase,G-protein, phosphoinositol, protein kinase C enzyme, inositoltriphosphate, diacylglycerol, intracellular calcium level, intracellularcalcium ion level, c-myc, ras, c-fos, c-jun, NK-kB, EIAI, AP-1, COUP,TCF-1α, TATA, TAT element, oxygen radical, CREB, CREM, Platelet DerivedGrowth Factor (PDGF), Colony Stimulating Factor (CSF), Epidermal GrowthFactor (EGF), Insulin Growth Factor (IGF), cytosolic tyrosine kinase,src, Src Homology 2 (SH2) domain, Src Homology 3 domain (SH3),serine/threonine kinase, Mitogen Activated Protein Kinase (MAP Kinase),Cytokine Receptor Superfamily, Signal Transducers and Activators ofTranscription (STATs), JAJ1, JAK2, Tumor Necrosis Factor-Receptor 1signal Transducer TRADD, chemokines of Rantes, and MIP-Alpha, andMIP-Beta.

[0095] The level of a repair marker may deviate from the level presentin the cell during normal function, and when it does so deviate,cellular repair processes are activated. This deviation may be positiveor negative, depending on the disease state and the precise state ofcellular repair currently in progress. As discussed herein, the“intensity” of the repair marker will refer to the degree of deviationfrom the level during normal cellular function, without regard towhether the deviation is positive or negative. The use of repair markersin establishing dose and duration of therapy is a novel mode ofadministering a pharmaceutical agent.

[0096] As defined herein, a “universal marker” is a constitutivelyexpressed molecule transcriptionally activated by the respective nucleicacid universally in all disease states for which the nucleic acid isspecific. “Universal markers” are specific for each nucleic acidemployed. While the universal marker is the only molecule that is notinjury specific and has no therapeutic value, it is expressive of theevent and duration of the ongoing repair process. Transcriptionalactivation gets shut off with the establishment of the state of cure. Assuch, the universal marker does not get modulated unless there is adisease state and the respective nucleic acid has therapeuticspecificity. The universal marker carries a direct quantitativerelationship to the daily per kilogram body weight dose (DKGD) of thenucleic acid employed. A universal marker defined for the prototypenucleic acid (defibrotide) is vWAg. Other “housekeeping genes” relatedto particular nucleic acids can be selected as per the target cellinvolved from the respective “housekeeping genes.”

[0097] Clinical and clinical laboratory markers may be determinedthrough blood tests, urine tests, clinical observation or identificationof blood clots by any of several conventional techniques, as well as themore novel techniques of determining genomic transcriptional andtranslational activity by DNA finger printing, PCR and the like. Toevaluate the markers, the laboratory analyses measure levels of certainproteins, lymphokines, enzymes and relevant molecules. Clinical markersinclude blood pressure, visible tissue damage, signs of inflammation,ecchymoses, and the like. Clinical markers vary from one disease toanother. Moreover, like HIV, many diseases progress through severalclinical stages during the process of recovery. The clinical markers ofone stage of a disease are frequently different from the clinicalmarkers in other stages of the disease, befitting different stages ofthe pathogenic picture.

[0098] The detection of markers relevant to the particular disease,stage of that disease, and as baseline for dose escalation, must firstbe identified. Any observable characteristic generally accepted by theskilled practitioner as being associated with a specific disease statemay be employed as a clinical marker. See, e.g., Harrison's PRINCIPLESOF INTERNAL MEDICINE, 10th Edition, Petersdorf et al. Eds., McGraw Hill.The skilled artisan would readily recognize those markers indicative ofa pathological state.

[0099] One critical marker is chosen at each respective stage of therepair process and the maximum efficacious dose for that markerestablished. Administration of that dose induces correction of otherstage-specific markers not necessarily identified or aimed at duringtherapy (i.e., “stage specific pleiotropism”). Following normalizationof the first chosen marker, a second marker which continues to deviatefrom the normal condition is chosen. The dose that normalizes the secondmarker (i.e., the higher dose) is likely to further improve the firstmarker incrementally.

[0100] Initial administration of the selected dosage is followed byincrementally increasing dosages until the “maximum efficacious dose” isreached. A panel of laboratory assays to determine the state of themarkers (e.g., absence, increase, decrease) is repeated every 3 to 7days during therapy. These results together with the clinical markers ofdisease would indicate whether the defibrotide, or other nucleic acidderivative, is adequate in dose and duration to cause improvement in thepertinent marker or markers while simultaneously being totally safe toadminister. Therapy is continued with escalating doses over sufficienttime to assure complete normalization (i.e., the clinical laboratoryassays, when compared to the reference range, are indicative of thenormal condition) of the pertinent markers. When normalization isreached, therapy is stopped.

[0101] Although therapy is discontinued, the patient is tested weeklyfor the current state of the pertinent disease marker. If relapseoccurs, therapy is reinitiated at the highest dose level of the priorcourse of therapy until normalization is again reached. While optional,it is advisable to continue escalating the dose level to potentiallyreach a shorter duration of therapy.

[0102] The highest tolerable dose per day which is complication free(e.g., no bleeding, thrombopathy) is preferred since treatment periodsare usually shorter at higher dose levels. Therapy cycles are repeateduntil there is complete and irreversible normalization of the pertinentmarkers at which point the patient is cured. A marker is considered tobe irreversibly normalized if it remains normal for three months withouttherapy.

[0103] There is a certain dose level which will ultimately give plateaulevels on a particular marker, and irrespective of how long the doserange is continued, the level of the molecule will not go higher unlessthe dose (or cellular uptake of the respective nucleotide) is increased.This agrees with accepted biochemical knowledge, i.e., the more thenumber of receptors receiving signals, the more cAMP is produced and, asa consequence, the higher the transcriptional activity pertaining tovWAg is.

[0104] Minimum effective dosing is therefore counterproductive andmarkers have to be used to assess the maximum efficacious dose.Application of the higher dose will promptly lead to higher levels in ashorter time (high m-efficiency score). This is confirmed from thecellular uptake curves.

[0105] Once a plateau is reached with the maximum efficacious dose, them-efficiency score can thereafter be used along with the maximum highestlevels of the last day to assess how long therapy should be continued tocomplete the repair process, i.e., when the maximum efficacious dose iscontinued when m-efficiency score is less than 1.0, the nucleotide nolonger exerts any further therapeutic effect. This observation leads tothe statistical definition of “maximum therapeutic dose,” i.e., the timeslot of the total administered dose beyond which further repair of theselected marker would not take place at that particular dose level.

[0106] If another disease marker were selected, the maximum efficaciousdose and maximum therapeutic dose would be redefined for that secondstage marker.

[0107] One skilled in the art, based on the information presentedherein, would be able to detect and determine finer disease/repairmarkers so as not to miss complete cure. Any abnormality in any markershould prompt reinitiation of therapy, even if no visible diseasemarkers are observed, since many of the markers of the subclinical stagewill be biochemical molecules, e.g., an interleukin.

[0108] Treatment in Accordance with the Invention

[0109] A preferred embodiment of the treatment method according to thisinvention is diagramed in FIG. 1. An initial laboratory test panel(box 1) is first run which would consist of the respective set of“disease markers” and the universal panel of “repair markers” consistingof signal transduction/second messenger panel molecules. Additionallycertain laboratory assays are used to assure that the dosages are safefor the patient being treated. For defibrotide these may includeprothrombin time, activated partial prothromboplastin time, thrombintime, reptilase time, bleeding time, platelet function assays andcoagulation factors (see baseline coagulation panel). “Disease markers”are utilized to indicate the overall therapeutic efficacy of the doses.These markers may be identified through blood tests, urine tests,clinical observation or identification of blood clots by any of severalconventional techniques, or by the more refined techniques such as DNAfingerprinting and PCR. To evaluate the “disease markers” the laboratoryanalyses measure levels of certain proteins, lymphokines, enzymes andrelevant molecules. Clinical markers may include blood pressure, visibletissue damage, signs of inflammation, ecchymoses, and the like.

[0110] An initial bolus of defibrotide (box 2) or its nucleic acidcomponents is given intravenously over 15 to 30 minutes. Immediatelythereafter the patient is given the daily dose of 40-400 mg/kg bycontinuous infusion. Preferably, the initial dose is a bolus (25-50mg/kg) followed by 24-hour dose which is increased in 50 mg/kg/dayincrements every 1-3 days. The starting base-line dose may be from40-400 mg/kg/day depending upon physician preference and the respectivedisease state treated. Lower initial doses are preferred for thosetherapeutic compounds which enter the cell nucleus more readily and arethus effective at lower doses. The bolus and daily dose for chemicalderivatives of the nucleic acids may be calculated as a proportion ofthe defibrotide dose based on the relative cell-entry rate. It ispreferred to administer this dose intravenously using two IV bags of 50ml D5W, each bag infused over 12 hours. If for any reason the infusionis interrupted, the rate of infusion would be thereafter adjusted sothat the patient will have received the calculated 12 hour dosage at thecompletion of the specified time period. This 24 hour dose range canalso be administered in 2-4 bolus injections or per oral administration.

[0111] Defibrotide or other selected nucleic acid derivative may beadministered parenterally, orally or locally by application to the skin.Parenteral administration is in the form of continuous intravenousinfusion or intravenous bolus injection. Intravenous infusion may beaccomplished by gravity feed, pump delivery or other clinically acceptedmethods. Oral administration may include the use of vials, capsules,tablets or powders for any method of enteric administration.

[0112] To permit clinically practicable administration of defibrotide inthe amount necessary, materials for delivery of the agent optionallycomprise 2×50 ml D5W IV bags each containing one-half of the calculatedtotal 24 hour dose in milligrams of defibrotide, each bag infused over12 hours for the IV-continuous infusion at the maximum tolerable doses.Alternatively, the total 24-hour dose can be administered by bolusinjection every 8-12 hours. The initial bolus injection and thesubsequent outpatient bolus maintenance infusions are given, forexample, in 3×25 ml D5W bags, each bolus to be infused over fifteen tothirty minutes. The oral dosage outpatient maintenance therapy inmilligrams given daily (divided into 3-6 doses by mouth) would be themultiples of 2× the maximum tolerable IV dose.

[0113] The same dose is given for three days and the laboratory testpanel is repeated (box 3). A full coagulation profile and tests formarkers should be run before and after any dose escalation. These testsresults are compared with the initial test data to determine if any ofthe markers (which may include laboratory data or clinical observationfor the disease being treated) have changed. A change is expected tooccur in at least one marker within 3-21 days, indicating thatdefibrotide is having an effect. After each test the dose of defibrotideis increased by 50 mg/kg/day, dose for chemical derivatives beingproportional to the cell entry rate for the respective nucleic acid, andcontinued at that dose for three days before retesting. This pattern ofescalating the dose and repeating the laboratory panels is repeated(boxes 4 and 5) until the patient's “maximum tolerable dose” (MID) isreached or until the disease/repair markers have plateaued or completelynormalized.

[0114] If three consecutive values for a selected marker are about thesame, a plateau has been reached. This procedure is followed for aminimum of 21 days (box 6). Disease/repair markers are checked andcoagulation profiles are run on weekly intervals to monitor response. Ifno response is observed, i.e., no change in the level of any marker (box7), therapy is discontinued (box 8), and treatment is determined to havefailed. If, after 21 days (box 6), no plateau is reached, butimprovement in the disease markers has occurred (box 14), the dose maybe doubled or the MTD may be given (box 15).

[0115] If the markers are normal (box 9), therapy is discontinued (box10). Tests continue to be repeated weekly for up to three months, notingany change in markers that would indicate relapse. If no relapse hasoccurred and no new markers have appeared after three months (box 11),therapy is discontinued (box 12) and the patient is considered cured.Should an old marker reappear or a new marker appear (box 13), the lastprevious dose is doubled, and therapy is resumed at that dose level. Ifdoubling of the dose would exceed the MTD, the MTD would beadministered.

[0116] Selection of Markers

[0117] The correct identification of markers are based on theidentification of the pathways of disease pathogenesis and therespective repair processes and pathways. The mechanism of efficacy ofthe therapeutic nucleic acid simulate or are superimposed on thecellular pathways of the respective repair process they induce. Forexample, using defibrotide as the clinical agent, one would (1) identifythe known signal transduction systems and second messengers of therepair process, (2) define the most probable nucleic acid-induced repairmarkers of the known cellular repair pathway, and (3) define markers ofthe disease process related to disease pathogenesis.

[0118] Many disease processes are pathogenically based on overactivebody defense mechanisms. As such, a compound whose intracellularconcentration can be a repair marker in one disease state can be adisease marker in another disease state. In such a case, the markerwould usually be under-regulated by defibrotide instead of induced.Similarly, a marker of normal cellular function, if deficient, may be adisease marker. For example, the paralysis of cellular function of CD4cells by the HIV retrovirus is secondary to the compromise of normalcellular markers of transduction pathways and second messengers.

[0119] G-proteins instrumental in the activation of adenylyl cyclase arelikely to be deficient in their active form with a low dose thresholdlevel. In this case, the deficiency of the normal cellular marker ofG-proteins would be a disease marker. Since defibrotide affects theadenylate cyclase pathway (increased cAMP by defibrotide), defibrotidewould restore the second messenger of cAMP, which therefore would be arepair marker.

[0120] The maximum therapeutic dose in turn would again follow theguidelines described above for vWAg, since this universal marker willget elevated with modulation of any phase of repair process such as, forexample, receptor up-regulation, signal transduction or induction oftranslation and/or transcription, shutting off oftranscription/translation which in turn may happen by activation ofCREM, which is the inhibitor transcription factor of CREB, i.e., thelatter is cAMP dependent initiator of the transcription factor of theCRE which in turn is the portion of the DNA enhancer sequence responsiveto cAMP and cAMP associated transcription factors, such as c-mycproducts, c-fos products, ATP Activation Factor, Serum ResponsiveElement (SRE), API transcription factor (ATF), IV-Long Terminal Repeat(LTR), leucine zipper transcription factors of c-fos/c-jun. (ATF, SRE,AP1 sites in c-fos promoter/enhancer all respond to cAMP without therequirement of SRE. Protein Kinase A activates endogenous CREB activityand will enhance viral transactivation).

[0121] The prototype high molecular weight defibrotide, nativedefibrotide, low molecular weight native defibrotide, and chemicaldefibrotide derivatives regulate genes which are regulated by cAMP.These genes include vasoactive intestinal peptide (VIP), somatostatin,human chorionic gonadotropin, phosphoenolpyruvate carboxylkinase,tyrosine hydroxylase, fibronectin, prolactin, ornithine decarboxylase,interleukin-6 gene, c-fos oncogene, haptoglobin, hemopexin, C-reactiveprotein (CRP), as well as other cellular genes which are regulated bycAMP responsive element (CRE), transcriptional factors interacting withCREB (which is 43 kd protein that interacts with CRE via leucine zipper,such as c-myc products, c-fos products, ATP (Activating Protein), SRE(serum responsive element), API. Protein kinase A will activateendogenous CREB activity and will also enhance viral transactivation.CRE/CREB related transcription of genes including HIV Long TerminalRepeat (LTR) will be positively induced with high cAMP levels.

[0122] The selected nucleic acid, e.g., defibrotide, will affect onlyinjury-dependent parameters in each individual patient. As such, nouniform action will be observable in all patients. For the nucleotidetranscriptionally-activated parameters, analysis is made for the highestvalues in each dose range. For the nucleotide transcriptionally shut-offparameters, analysis is made for the lowest value in each dose range.

[0123] Therapy Based on Universal Marker

[0124] Several markers have now been shown to reflect transcriptionalgenomic activity by nucleotides which increase cAMP, adenylate cyclasevia the interaction of G-proteins, and phosphorylate transcriptionalfactors via protein kinase A. Such markers include von Willebrandtantigen (vWAg), tissue plasminogen activator antigen (AgTPA) andβ₂-microglobulin. While vWAg, AgTPA and β₂-microglobulin arerepresentative markers, any molecules which are initiated bynucleotides, or derivatives such as defibrotide, to inducetranscriptional activity are included.

[0125] It has been discovered that vWAg may be employed as a universalmarker to guide the assessment of the duration of therapy, i.e., themost therapeutic dose, as well as the most efficacious daily dose. Theinventors have discovered that vWAg is transcriptionally activated bydefibrotide irrespective of the type of injury. Analysis of patient datahas led to the unexpected finding that with the onset of cure, vWAglevels decline. The production of vWAg will be activated by defibrotideonly for the duration of the injury and the repair process. In thisregard, defibrotide will not effect vWAg levels in healthy individualsor following the establishment of cure, i.e., vWAg level will decline tobaseline regardless of ongoing therapy. Concurrent analysis of vWAg withvarious “disease markers” correlated with changes in the disease markerlevels. In other words, it has been discovered that therapy dependentabsolute changes in disease markers (decline or increase) correlate withpeak vWAg levels. The normalization of disease markers, in turn,correlates with decline in vWAg levels.

[0126] vWAg is classified according to this invention as being auniversal dose marker. vWAg can be utilized as the universal marker forall nucleotides that induce activation of cAMP and protein kinase Aenzymes. vWAg is a plasma glycoprotein having a molecular weight ofapproximately 200,000 which is constitutively secreted by theendothelial cell. It is important in hemostasis as a prothromboticfactor (factor VIII/vWAg protein) and as an inducer of adherence ofplatelets to the exposed subendothelium. In every disease state, vWAglevels go up with increasing defibrotide dose levels when the dose isadequate to stimulate vascular endothelial function.

[0127] In accordance with the invention, an increase in the vWAg levelcorresponds to the induction of transcriptional activity of this gene bythe nucleic acid. Elevation of vWAg is representative of the ongoingrepair process. The decline in the level and eventual normalization ofvWAg during therapy is representative of the cure process. Plateau inthe level of vWAg correlates with the application of the maximumefficacious dose. Without exception, the elevation in the level of vWAgis concurrent with modulation of the disease marker and activation ofthe repair marker. Here the maximum efficacious dose is determined alongwith vWAg, so as to normalize the levels of these molecules between65-150%, and eliminate the intracellular oxygen radicals (measured bychemiluminescence, normal state being negative). For the prototype drug,defibrotide, the maximum efficacy in inducing transcriptional activationof vWAg occurs at doses of 40 DKGD and above, ideally within the DKGDrange of 40-400. The universal marker vWAg dose levels arerepresentative dose levels by the prototype'stranscriptional/translational modulatory effects. Fitting the definitionof universal marker, vWAg does not contribute to the expected correctionof bleeding time but acts as a functionally dormant molecule.

[0128] Another option is to empirically repeat therapy after three weeksfollowing cessation of therapy on the above principles. In this regard,the half life of the nucleic acid appears to be about three weeks, basedon the observation that universal marker vWAg requires 2-3 weeks to comedown to baseline levels with cessation of therapy. If the universalmarker vWAg is elevated during therapy with the previous maximumefficacious dose, there is still a lesion to treat, irrespective of thefact that there are no known or visible clinical, and/or documentedbiochemical repair or disease markers.

[0129] Therapy, in accordance with the invention, is geared to continueuntil vWAg is normalized while on established maximum effective dose.Thereafter therapy is discontinued and the same cycles are repeateduntil the maximum efficacious dose therapeutically initiated no longerinduces any elevation in vWAg, as would be observed in a normal healthyindividual.

[0130] Statistical Analysis

[0131] A statistical model has been used to assess the dose and durationof therapy with the ultimate objective of irreversible cure. For eachmolecular marker, calculations are presented, based on analysis of thedata from all the patients for the “first day value,” the “last dayvalue,” the “highest value” or “lowest value” (i.e., fortranscriptionally activated molecular markers and for transcriptionallyinhibited molecular markers, respectively), the “m-efficiency score,”and the “time required to reach the optimal effect of the nucleotide” atthe dose ranges employed. The “first day value” at a particular dose isthe “last day value” of the preceding dose range. The “minimum ofincreasing values” has been found to be the best parameter to follow fortranscriptionally turned on molecules while the “maximum of the lowestvalues” has been found to be the best parameter to follow fortranscriptionally turned off molecules.

[0132] The best parameter to follow the dose related induction oftranscriptional activity is the “minimum values of the increasinglevels” obtained on the first day of the initiation of each dose range.“Highest or increasing levels” represent the increase in level of amolecule whose production (transcription) is turned on with increasingdose levels. Choosing the minimum increase in the level in thetranscribed genome among all patients treated in any dose range enablesthe prediction of the worst performance with that dose of thetherapeutic compound. This enables the treatment of the worst performer,which allows turning on the genomic transcriptional activity in thegreatest number of patients within each respective dose range. Increasein the marker, as shown by “minimum highest value” represents that therepair process is ongoing, that is, repair molecules are being producedand transcriptional activity is ongoing.

[0133] The quantitative relationship between vWAg level and daily doseof the therapeutic compound is best visualized when the minimum value ofthe increasing vWAg levels in the population are analyzed (i.e., theworst performance levels in any one patient at any one dose range,“worst performance” implying that increasing the dose will incrementallycontinue to elevate the vWAg, which is biologically interpreted asmeaning that there are more repair events to go through).

[0134] Minimum increasing value is the parameter to use to confirm theevent of ongoing cellular repair. Maximum increasing value is thestatistical parameter to use to follow the completion of the repairevent. Maximum therapeutic dose is the dose at which vWAg on continuedtherapy will decline to a normal level.

[0135] The “maximum values of the lowest levels” obtained among allpatient data on the last days of treatment at each dose range aresimilarly used to analyze how increasing dose ranges affect thetranscriptional activities involving the “turned on” molecules. Thelevels will show progressive declines, i.e., the progressive turning offof the repair process with the onset of cure, in spite of the higherdoses.

[0136] Once maximum stimulation takes place (as assessed by the use offirst day minimum highest levels), the cell gets turned off. By the useof the maximum lowest levels of the last day, therapy is continued atthat particular dose level until these levels return to the baselinelevels on therapy, i.e., until there is no more ongoing transcriptionalactivity, i.e., the repair process is completed.

[0137] The m-efficiency value is the ratio of the respective elevatedlevel over the time taken for elevation to occur. The higher the dose,the higher the value of the numerator and the higher the m-efficiencylevel. Alternatively, the shorter the time (denominator), the higher isthe m-efficiency value.

[0138] Method of Treating HIV-Infected Patients with Defibrotide or ItsNucleic Acid Components

[0139] Defibrotide or its nucleic acid components modulate cellfunctions at the nuclear genomic level through one or more pathways bymodulation of the cell's genetic material, i.e., DNA itself ortranslation or transcription of the genetic information. Defibrotide orits nucleic acid components-induced cellular modulation restores thenormal functions of the cell such as the production of normal proteinsneeded by the cell and, in the case of HIV, the correction of theeffects of the abnormal, viral encoded genetic material by inhibitingits further production at the expense of the normal, virus-free geneticmaterial. In the course of the multiphase treatment, defibrotide or itsnucleic acid components is administered at dosages much greater thanpreviously described in the literature for other disease states. Thedosages and durations of the phases of therapy are adjusted according tothe results of laboratory studies performed on the patient's infectedcells. Preferably, in treating HIV, an initial bolus dose of 100 mg/kgin 50 ml DSW is infused over a period of 30-60 minutes followed by 200mg/kg/day infused in 250-500 ml DSW over a period of 3-24 hours. Fromday 2, dose is escalated to maximum tolerable dose, maximum efficaciousdose and maximum therapeutic dose levels. In this way, the HIV virus maybe inactivated and its proliferation arrested. Therefore, the progressof the disease may be arrested or ameliorated.

[0140] Because HIV virus adversely affects the genetic material andfunction of the cells, defibrotide or its nucleic acid components caneffectively treat HIV infection as long as the carrier CD4′ cell and/orthe monocyte harboring the virus preserves the physiological ability torevive itself. Therapeutic success with defibrotide, however, isstrictly dependent upon the assessment of the correct treatment dosesfor the respective disease states. Moreover, since the optimum functionof the normal cell, by definition, would not be compatible with anycomplications, defibrotide or its nucleic acid components at any definedmaximum efficacious dose, specific for any patient and disease statewould be complication-free.

[0141] Sarin et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85:7448-7451),as well as Leonetti et al. (Bioconjugate Chem., 1990, 1:149-153), haveshown that anti-sense oligonucleotides are potent inhibitors of HIV-1replication in cell culture. The methylphosphonate linkedoligonucleotides were found to be superior in this effect over thephosphodiester linked oligonucleotides, apparently as a result of theirresistance to nucleases. This property was deemed to be the factor inthe superiority, since oligonucleotides less than 20 bases in lengthproved to be ineffective inhibitors.

[0142] Efficacy of defibrotide or its nucleic acid components may haveseveral concurrently active mechanisms. Defibrotide or its nucleic acidcomponents may provide anti-sense neutralization of the viral proteins.Defibrotide's mechanism of efficacy may be at the nuclear level bymodulation of genetic functions via other pathways as well.Defibrotide's actions may be more apparent during viral phases whichinvolve translation and/or transcription of the DNA message, so as torevive the normal function of the cell at the expense of thedisease-specific molecules. This action may be analogous to anti-viraleffects of Ampligen (a mismatched double stranded RNA-molecule).However, whereas Ampligen exhibits immunostimulating effects, agentssuch as defibrotide are both immunostimulants and immunosuppressants.Defibrotide or its nucleic acid components may modulate viralpenetration into the cell via its known action of inhibitingintracellular calcium mobilization. Also, defibrotide or its nucleicacid components may directly inhibit viral enzyme reverse transcriptasevia inducing ATP production analogous to ddI (dedeoxyinosine), by virtueof its known action of inducing high energy metabolites (ATP, ADP,NADP/NADPH), possibly via modulation of Complex-I respiratory molecule.Defibrotide or its nucleic acid components may inhibit protein kinase C,analogous to Hypericin. Defibrotide may also decrease Tissue NecrosisFactor (TNF), a cytokine known to promote HIV activation, by its knowneffect on increasing cAMP levels at the correct defibrotide dose levelsimilar to Pentoxyfilline.

[0143] Whatever the mechanism, zinc is known to have an inhibitoryeffect upon nucleases acting on phosphodiester linkages, as well as anenhancing effect on base pairing. U.S. Pat. No. 3,770,720, teaches thatin the production of defibrotide, zinc should be removed from themolecule. However, in the treatment of AIDS, it is preferred that zincbe present. Moreover, it is preferred that iodine should also bepresent. In the defibrotide used in the Examples iodine was present inan approximate ratio of one zinc atom per iodine atom and a two to oneratio of zinc + iodine to nucleotide base.

[0144] As can be seen from comparing the cellular uptake data shown inFIGS. 2 and 3 with the data shown in FIGS. 4, 5 and 6, a greater levelof defibrotide enters the lymphocytes when biotin is present. Horn etal. (Plant Physiol, 1990, 93:1492-1496) has observed that biotinylatedmolecules enter the cell via the folate endocytic pathway. The data ofFIGS. 2-6 read in conjunction with the above-cited Horn reference,indicate that defibrotide with biotin may also use the folate endocyticpathway.

[0145] Defibrotide may jointly and/or selectively modulate one orseveral pathways. This modulation will be, only to the appropriatedegree thus surpassing all of the other anti-HIV agents in its lack ofside effects, yet presence of proven efficacy. Defibrotide will achievethis result only when the dose levels are tailored to the patient, stageof disease activity and/or reigning stage of viral activity.

[0146] The method of treating the HIV-infected patient begins with apanel of laboratory studies which include the quantitative evaluation ofthe activated peripheral blood mononuclear cell subsets, circulatingviral proteins, cytokinases and soluble cell-surface receptors. Thereare no patient inclusion or exclusion criteria for therapy. Patients inany or all of the four clinical stages of HIV-infection includinghistory of exposure (i.e., HIV⁺, Pre-ARC, ARC, and AIDS) are candidatesfor therapy. The initial administration of a selected dosage ofdefibrotide is followed by incrementally increasing the dosage ofdefibrotide until a maximum tolerable dose is reached. The laboratorypanel is repeated weekly during this therapy. These results togetherwith the clinical markers of disease would indicate whether thedefibrotide is efficacious and whether defibrotide should be continuedto be given alone or with other therapeutic agents.

[0147] The details of treatment and the dose ranges fitting the variousstages of the HIV disease will be expressed by retrospective analysis ofrespective laboratory and clinical markers. Additionally, dosage levelsand frequencies as well as the use of other anti-HIV medication willalso depend upon the individual patient or stage of disease and/or otherconcurrent medical conditions.

[0148] Before the initiation of therapy and weekly thereafter blood isdrawn from the patient and subjected to a panel of tests whichpreferably include activated peripheral blood mononuclear cell subsetsby two-color flow cytometry, lymphokines and soluble cell surfacereceptors by ELISA, and HIV-viral proteins by Western blot analysis. Theperipheral blood mononuclear cell subset analysis will usually includeeither CD4⁺, CD8⁺, CD19⁺, CD25⁺, CD56⁺, and HLA-DR alone, combined withone another, or combined with the quantification of monocytes. TheWestern blot protein tests include gp-24, gp-17, gp-120 and gp-160. TheELISA test measures TNF, sIL2R, sIL1 and soluble CD8. Every third week,it is preferred that cell cultures for HIV antibody neutralization, PCRand reverse transcriptase determinations be made.

[0149] HIV-I Gene Therapy in Accordance with the Invention

[0150] Gene delivery thus far has been a method by which foreign geneticmaterial is introduced into a suitable target cell usually via viralvectors. Such strategy generally consisted of an ex vivo and an in vivophase. In the ex vivo phase the foreign gene is inserted into targetcells derived from the recipient. The engineered cells containing thenewly inserted gene are expanded ex vivo. In the in vivo phase, theexpanded engineered cells are transplanted into the recipient.

[0151] This modulatory therapy is the first of its kind which managestherapy from cell surface signaling to genomic modulation utilizing theoral and/or intravenous administration of nucleotides, without utilizingretrovirus, adenovirus or other gene viral vectors traditionallyemployed in gene therapy. Gene therapy has not, heretofore, beenutilized without cellular transfection with viral vectors, and neverbefore by oral or intravenous administration of nucleotides to humans.

[0152] Gene therapy has not, heretofore, been tried without theinteraction of viral vectors, i.e., by the administration of nucleicacid-based pharmaceutical agents orally and/or by intravenous route. Theprototype drug defibrotide, although administered to patients over thepast 5-6 years, has never heretofore been contemplated for gene therapy.In addition, in other modalities of gene therapy, dosage has never beenassessed by molecule markers. Molecule markers have never been definedwithin the system of secondary messengers, signal transduction systems,promoters (DNA sites which are on the same chromosome as the genetranscribed and to which RNA polymerase binds), enhancers (DNA regionsthat control a promotor from a great distance, sometimes as much as30,000 bases), and transcription factors (diffusible regulatory proteinswhich bind to DNA transcription activation domains and regulate the rateof transcription by RNA polymerase).

[0153] HIV-disease has not been previously interpreted as a disease ofdismodulation involving the genomes, cellular secondary messengers andcellular signal transduction systems. The specific pathways affected bythe HIV-retrovirus have not been clearly delineated. Therapy ofHIV-disease has not previously attempted to reclaim the affectedcellular function systems from the virus by reversing the dismodulationat the various levels by using exogenous therapy involving variousmodulators of these systems.

[0154] The therapeutic approach of the invention disengages itself fromthe common practice of planning therapy based on clinical staging. Theplanning of therapy is based on the identified mismodulations of (a)membrane lipids and cytoskeleton; (b) cell-surface receptor/ligandinteractions; (c) secondary messengers; (d) signal transducers; (e)cellular transcription factors utilized in viral replication: as well asbased on the identified (f) oncogenes; (g) viral transcription factors;and (h) viral genomes. The method of therapy disclosed herein for HIVmay also be used in treatment of other viral infections and neoplasms.

[0155] These mismodulations are classified into marker categories of (I)repair markers (items a-e) and (ii) disease markers (item f-h). Theobject of therapy in accordance with the invention is to (I) reestablishrepair markers at the constitutively expressed tissue levels; and (ii)eliminate disease markers (in case of the oncogenes to reverse thetransformation).

[0156] Irrespective of disease stage or clinical status the patient isscreened with the complete panel of secondary messengers and signaltransducers (repair markers), since all repair markers are biochemicallyinterdependent. Repair markers reflect the underlying logic oftranscriptional regulation. Therapy is aimed to concurrently induce somemarkers and suppress other markers. The prototype nucleotide if used atthe correct doses (which are guided by the respective repair markers)can accomplish this goal.

[0157] Elimination of disease markers by the therapeutic nucleotidecompound will occur at various levels. It can be an indirect phenomenonbased on modulations of secondary messengers, such as cAMP; it can be adirect phenomenon based on modulations of the phosphorylation eventsinvolving genes and transcription factors. For example, cAMP activatesprotein kinase A enzymes, Ca²⁺ activates protein kinase C enzymes, theprototype nucleotide up regulates cAMP, and downregulates Ca²⁺, or itcan be a direct phenomenon based on modulation of cAMP responsive genepromoters (CREM, as enumerated above).

[0158] While not being bound to any specific mechanism of action, thefollowing are proposed.

[0159] Proposed Mechanism A. Induction of sIL2R gene and HIV-1 LTR areinterdependent phenomena. If the protein kinase C dependent sIL2R geneis turned off by high cAMP levels, activation of HIV-I LTR isconcurrently suppressed as well.

[0160] Proposed Mechanism B. Increased cAMP levels have been shown toinduce viral replication (Nokta and Pollard, 1992, AIDS Research andHuman Retroviruses 8(7): 1255-1261). HIV-I REV/ENV genes are bothphosphoproteins. There may be other routes for cAMP-induced replicationof HIV-I. Although administration of the maximum efficacious dose willincrease cAMP levels, prolonged administration of the nucleic acid atthe maximum efficacious dose, so as to realize the successfuladministration of the maximum therapeutic dose would culminate indeclining cAMP levels, since vWAg decreases on therapy if and once themaximum therapeutic dose is administered. Hence administration of themaximum therapeutic dose is paramount in overcoming cAMP induced viralreplication. This phenomenon may at least partially, be based on theinduction of protein kinase C, as a secondary biochemical event (i.e.,protein kinase C induces sILR2 gene, which in turn modulates proteinkinase C so as it can directly inhibit cAMP).

[0161] Proposed Mechanism C. The transcription factor NF-kB binds toboth the HIV-I enhancer, and the sILR2 gene. Protein kinase Cphosphorylates its inhibitor IkB and releases active NF-kB. IncreasedcAMP levels by inhibiting directly the Ca²⁺ induced activation ofprotein kinase C would modulate this phosphorylation event, anddownregulate the transcriptional activities related to NF-kB. SinceNF-kB binds to both the HIV enhancer and IL2 receptor, increased cAMPlevels will downregulate HIV-I replication.

[0162] Proposed mechanisms B and C show that increased cAMP levels canbe both deleterious and beneficial. It can be clearly seen that theprototype nucleotide is an overall “downregulator” of biochemicalevents, if maximum therapeutic and efficacious dose levels areadministered.

[0163] It has also been discovered that the co-administration of varioussequence specific, anti-sense or missense nucleic acids with, forexample, defibrotide, would (1) alleviate the complication of cAMPinduced viral replication; (2) induce inhibition of viral replicationmediated via modulations of cAMP, protein kinase A, protein kinase C,cellular redox state, G-proteins, or cAMP induced gene promoters (inthis regard, defibrotide and other nucleotide derivatives introduce forthe first time into anti-HIV therapy nucleotides with no sequencespecificity that concurrently modulate the totality of the cellularsecond messenger/signal systems for rapidly transducing extracellularsignals into specific patterns of gene expression in the nucleus); (3)concurrently induce inhibition of viral replication with sense,anti-sense, or missense nucleic acids (e.g., DNA, mRNA, DNA/RNAribosomes, inhibitors of viral protease, viral integrase); and (4)introduce a modality of gene therapy (i.e., genetic engineering) whichcan be safely administered to humans, which does not utilize viralvectors, which can be administered either intravenous or orally, whichenables administration of sequence specific combination of nucleic acidsadjusted specifically to the selected parts of the HIV-genome andcellular repair pathways, which adjust the dose so as to modulateselected genes or cellular/viral molecules, which enables the mostefficient administration of various different nucleotides with differingcellular uptake dynamics and chemical anti-viral potencies, and whichadministers excess DNA to enable the self-integration of DNA.

[0164] This process is superior to present viral vector directed genetherapy and would also enable competitive inhibition of proviralintegration, and/or dislocation of the integrated pro-virus. Cellularuptake dynamics would directly define the anti-viral and geneticmodulatory capacities of each respective nucleotide. Nucleic acidderivatives having chemical modifications are as described previously(e.g., nucleotides conjugated with poly(L-lysine) or which is modifiedby, for example, the addition of amino acids such as lysine, histidineand arginine, the addition of optimum concentrations of folate and/orbiotin, the addition of the optimum ratios of metals and ions includingzinc, manganese and iodine, by the addition of 5′-polyalkyl moieties,cholesterol, vitamin E, 1-2-di-O-hexadecyl-3-glyceryl and otherlipophilic moieties and/or modified by the replacement of phosphodiesterbonds with phosphothiotate bonds) and combination nucleic acids would beemployed.

EXAMPLES Example 1

[0165] To measure the effect of defibrotide on HIV it was firstnecessary to label the drug and determine whether defibrotide will enterthe nucleus of the human cell. Knowing the phosphodiester linkages indefibrotide, its comparative nuclear penetration was assessed bylabelling defibrotide with a photo-activatable analogue of biotin. Thebiological activity of defibrotide after labelling was considered tohave been preserved since published data shows that previousoligonucleotide probes have been labelled with conjugates and stillremained biologically active. Image analysis utilizing a cold CCD camerarevealed that uptake of defibrotide was localized in the nucleus. Thissupports the hypothesis that the mechanism of efficacy for defibrotideis largely contributed to by its modulatory activity on the geneticmaterial of the cell, no matter what disease entity is being treated. Asshown in FIGS. 2 and 3, the nuclear uptake of defibrotide is directlyproportional to the concentration of defibrotide with biotin. Theobserved uptake supported the increased efficacy of defibrotide with thelarger doses used, and also supports the hypothesis that at criticallyhigh dose levels various previously unknown different effects ofdefibrotide can be seen. It was also observed that uptake by monocyteswas significantly greater than that by lymphocytes.

[0166] The cellular uptake of defibrotide without biotin and labelledwith cyanine dye Cy5.18 was also measured. It was observed thatbiotinylation of defibrotide enhanced the cellular uptake of defibrotidein the lymphocyte population. However, there was no difference in uptakebetween monocytes incubated with biotinylated or fluorescently taggeddefibrotide. This can be seen by comparing FIGS. 4 and 5.

Example 2

[0167] To further confirm the specificity of defibrotide for thetreatment of HIV infection, HIV infected peripheral blood mononuclearcells with varying doses of defibrotide were evaluated by staining forall viral envelope proteins using concanavalin A (Con-A) stimulated andunstimulated cells (Anti-HIV 1, and Anti-HIV 3 specific Anti-HIVantibody). The blood sample was obtained from a patient using anevacuated blood collection tube containing sufficient EDTA to preventcoagulation of the sample.

[0168] Mononuclear leukocytes (white cells) were obtained by layering a1:1 (volume:volume) blood to RPMI 1640 tissue culture medium (GrandIsland Biological Co.) aliquot over histopaque (d=1.077, Sigma ChemicalCo.) under sterile conditions. The white cell population was suspendedin a solution of the RPMI 1640 medium supplemented with 10%heat-inactivated fetal calf serum and gentamicin, at the concentrationof 5 micrograms/milliliter. The white cells were then concentrated to alevel of two million cells per three milliliters (2×10⁶ cells/3 ml) ofthe above solution. The white cells were collected in flat-bottomedmicrotiter containers (Cell Wells, Corning).

[0169] The cell populations were further divided into two groups. Onegroup received stimulation by Con-A, the other group remainedunstimulated by Con-A. Con-A stimulation enhances the uptake of theantibody-dye label by HIV-contaminated cell components, therebydemonstrating an increase in the expression of the HIV protein.

[0170] Subpopulations of unstimulated and stimulated white cells werethen incubated in the presence of discrete concentrations ofdefibrotide. Each successive assay employed successively greaterconcentrations. A control sample of incubate containing no defibrotidewas also prepared. A labelling antibody solution was prepared bydirectly conjugating Cy5.18 with human α-HIV antibody to a finaldye/protein ratio of 5.0 (α-HIV-Cy5.18).

[0171] The cell subpopulations were again divided into two groups, onegroup for intracellular antibody labelling, and one group for surfaceantibody labelling. Cells reserved for intracellular labelling werefixed with 70% ETOH, washed twice with monoclonal wash, and thenresuspended into a solution containing 200 microliters of Hank'sbalanced salt solution (HBSS), supplemented with 2% FCS and 0.1% sodiumazide (monoclonal wash) and 5 microliters of α-HIV-Cy5.18 solution. Theentire cell preparation was incubated for 45 minutes at 4° C. The cellpreparation was then washed twice with the monoclonal wash, andresuspended in 1% paraformaldehyde.

[0172] Cells reserved for surface labelling were prepared by washingtwice in monoclonal wash to which 5 microliters of α-HIV-Cy5.18 havebeen added. Next, 20 microliters of specific surface glycoproteinmonoclonal antibody was added to the incubation solution. The surfaceglycoprotein antibody solution contained CD3-FITC (heterogenous T-cellantibody conjugated with fluorescein isothiocyanate dye) and CD4-RPE(helper T-cell antibody conjugated with phycoerythrin dye) obtained fromBecton-Dickinson.

[0173] All cells thus prepared were then analyzed using aBecton-Dickinson FACS 440 dual laser (argon/krypton) flow cytometer. Theexpression of HIV proteins was determined on a per-cell basis.Fluorescence was measured on a logarithmic scale but converted to alinear scale for analysis.

[0174]FIG. 7 shows HIV protein expression at selected dosages. Assayresults for the same sample shown in FIG. 8 are in terms of theintensity of the fluorescence of certain antibody-labelled mononuclearleukocytes (Mean Linear Fluorescence Intensity). Fluorescence intensityis proportional to HIV protein expression, and thus the activity of HIV.It is seen that the expression of the HIV proteins decreases and thenlevels off with increasing concentrations of defibrotide.

[0175] Before administration of defibrotide, Con-A stimulated cellsexpressed 32% more viral proteins. However, after administration of 20mg of defibrotide, both stimulated and unstimulated cells express 70%less viral proteins. At 30 mg concentration of defibrotide in both Con-Astimulated and unstimulated cells the expression of viral proteinsleveled off. This supports the specificity of defibrotide for HIV-virusas well as the fact that if cells are induced to divide, translatinginto proliferation of the virus, more HIV virus can be killed, albeit,at higher doses.

Example 3

[0176] Patients with various diseases of vascular prothromboticbackgrounds were treated with escalating dose levels of defibrotide. Avariety of coagulation and hematological assays with other molecularmarkers of inflammation, etc., were conducted on blood samples drawnfrom the patient before and after each dose escalation. From an analysisof the test results and clinical observations, it was discovered thatcertain effects of defibrotide lead to a remission state of certainspecific aspects of disease states corresponding to the various doseranges employed.

[0177] As an example, hematological recovery in thromboticmicroangiopathy, generally, yet not exclusively, occurred when thepatient received doses of defibrotide ranging from 20 to 30 mg/kg/day.These doses however did not cure the renal lesions since creatininelevels remained above normal (or only partially corrected) at the doselevels where hematological recovery was complete. Renal recoveryevidenced by normalization of creatinine levels occurred between 40 and250 mg/kg/day.

[0178] Even in the presence of normalization of creatinine levels (theconventional criteria of complete recovery) it was observed thatcomplete remission was yet to be reached by the observation of elevationof blood pressure, low AgTPA and high fPAI levels. Therefore, doses ofdefibrotide continued to be increased until blood pressure levels becamenormal. The dose elevation not only treated blood pressure, but also ledto further improvement of creatinine. Thus, treatment withmarker-dependent doses, applied correctly, led to a state of “cure”.

Example 4

[0179] In a normal individual, increasing the DKGD dose does not induceany elevation in the vWAg since there is no ongoing repair process,i.e., no disease oral/pharyngeal candidiasis, polyarthralgias andtuberculosis was treated with defibrotide.

[0180] On Day 1 of treatment, a 360 mg/kg IV bolus of defibrotide wasadministered. Thereafter, a dose of 160-275 mg/kg/day was administered.Defibrotide was administered 86 days out of a 118 day treatment course.

[0181] Progressive increase in weight and amelioration of diarrhea wasobserved throughout the therapy period, a total weight gain of 12 kgoccurring during the treatment period. Improvement in Kamofskyperformance score started at day 3 and increased from a score of 3 to ascore of 10 over the treatment period.

[0182] The effect on arthralgia was observed by the third consecutiveday of treatment and was found to be strictly dose dependent. Uponcessation of therapy arthralgia relapsed to original condition andentered remission upon reinitiation of DNA therapy.

[0183] The effect on Herpes began on day 4 of treatment. By day 36 ofthe treatment period, genital Herpes lesions were in complete remission.By day 68, Herpes labialis lesions were in complete remission. Norelapses were seen with temporary cessation of defibrotide.

[0184] Tables I and II summarize pertinent laboratory markers. TABLE IAb- Ab Ab- TIME solute solute solute B₂-micro- (days) Lymphocyte CD4 %CD4 CD8 % CD8 globulin 1 1388 13.2 183 22  305 N.D. 26 1152 32.0 369 50 576 N.D. 90 N.D. N.D. N.D. N.D. N.D. 3582 104 N.D. N.D. N.D. N.D. N.D.1348 118 3300 21.0 693 32 1056 N.D.

[0185] TABLE II TIME (days) IL2 TNFα IL6 cAMP cGMP cGMP/cAMP 8 14.3 30.141.6 2.0 1.03 0.52 76 7.3 14.7 3.7 3.65 2.10 0.58

[0186] Elevated cAMP/cGMP was observed at the onset of therapy,signifying activation of both protein kinase A and protein kinase Cpathways. A progressive rise in absolute and T lymphocyte numbers, CD4and CD8 was seen. A decrease in IL1, IL-2, IL-6 and TNF-α was observedduring treatment.

[0187] Complete remissions in accompanying disease states include Herpeslabialis, oropharyngeal candidiasis, arthralgia, and Herpes genitalis aswell as accompanying tissue damage. Complete normalization of TBfindings (Chest x-ray) with apparent radiological remission occurred.

Example 6

[0188] A 25-year old white HIV⁺ female was treated with defibrotide. Atthe onset of therapy, the patient was asymptomatic but had a low CD4count.

[0189] On day 1 of the treatment, a 200 mg/kg IV bolus of defibrotidewas administered. Thereafter a dose of 150-275 mg/kg/day wasadministered. Anabolic effects of the DNA were seen by day 13.

[0190] DNA therapy was terminated after 29 days secondary to a rise ofCD4 percent and absolute counts. DNA therapy was reinitiated 25 dayslater secondary to a decline in CD4 percent and absolute counts. Therapywas continued on an outpatient basis, intravenous administration beingalternated with oral administration.

[0191] Tables III and IV summarize pertinent laboratory data. In thispatient, all tested interleukin levels were normal. TABLE III Ab- Ab-TIME Absolute solute solute β₂-micro- (days) Lumphocytes CD4 % CD4 CD8 %CD8 globulin TREATMENT CYCLE #1 1 973 15.2 148 20.1 196 N.D. 28 110048.0 528 50.0 550 3300 TREATMENT CYCLE #2 1 429 34.0 146 22.0 94 N.D. 201945 15.0 292 20.0 389 2468 56 2600 29.0 754 20.0 520 N.D.

[0192] TABLE IV TREATMENT CYCLE #2 TIME (days) cAMP cGMP cGMP/cAMP 11.25 0.98 0.78 8 1.55 3.00 1.94 20 1.50 3.40 2.27

[0193] Treatment was characterized by increases in CD4, CD8, totallymphocyte, total T-lymphocyte counts accompanied by elevations in cAMPand cGMP, and in therapy related decreases in IL-6 and TNF-α. A totalweight gain of 7 kg was observed.

Example 7

[0194] A 33-year old white male with AIDS and opportunistic infectionsincluding Herpes labialis associate with necrotic lesions,oral/pharyngeal candidiasis, tuberculosis and crytococcal diarrhea.

[0195] On day 1 of treatment, a 200 mg/kg IV bolus was administered.Treatment at a dose of 100-250 mg/kg/day was continued until day 40. Thelower doses being given on days 7-13 having been reduced secondary toprolonged APTT. Treatment was thereafter discontinued due tounavailability of the drug. The patient died 8 days following cessationof therapy.

[0196] An anabolic effect was seen from day 6. Diarrhea was controlledfrom day 3 and cultures for cryptococcus became negative on day 15.Lesions of the lip began healing on day 5 and were completely healed byday 18. Odynophagia improved from day 5. Performance score beganimproving by day 3, reaching an optimum level of 5 between days 16 and21.

[0197] Tables V and VI summarize pertinent laboratory data. TABLE V TIMEAbsolute Absolute Absolute (days) Lymphocytes CD4 % CD4 CD8 % CD8 7 70010.0 70 29.0 203 18 800 10.0 80 17.0 136 44 700 8.0 56 14.0 98

[0198] TABLE VI TIME cGMP/ (days) IL1 IL2 TNFα IL6 cAMP cGMP cAMP 21 10518.0 95.1 40.6 1.4 1.1 0.79 33 85.5 7.6 14.8 45.1 1.5 0.96 0.64

[0199] Decline in elevated IL-1, IL-2 levels and complete normalizationof TNF-αlevels was observed. An increase in Il-6 was seen with cessationof therapy. At the time of death, a 3 kg weight gain was observed, andHerpes labialis and oral/pharyngeal candidiasis were in completeremission.

1 19 15 base pairs nucleic acid single linear 1 GGTTGGATTG GTTGG 15 15base pairs nucleic acid single linear 2 GGTTGGATCG GTTGG 15 15 basepairs nucleic acid single linear 3 GGATGGATCG GTTGG 15 15 base pairsnucleic acid single linear 4 GGTGGTGGTT GTGGT 15 18 base pairs nucleicacid single linear 5 CAGCTGCACC TGCCAAGC 18 19 base pairs nucleic acidsingle linear 6 ATAAAATATA CCATATACA 19 21 base pairs nucleic acidsingle linear 7 TCATAAAATA TACTATATTC A 21 30 base pairs nucleic acidsingle linear 8 ATATTAAAGA ACGCTGTTTA CAATACTTGG 30 18 base pairsnucleic acid single linear 9 ATGCAGTTGT GAAGAGAA 18 33 base pairsnucleic acid single linear 10 AATTAAGGCA TAAGAAAACT AAGAAATATG CAC 33 30base pairs nucleic acid single linear 11 TCTCTCCCTC AAGGACTCAGCTTTCTGAAG 30 18 base pairs nucleic acid single linear 12 CAATAATAAAAGGGGAAA 18 21 base pairs nucleic acid single linear 13 AGTGCAACCGGCAGGAGGTG A 21 40 base pairs nucleic acid single linear 14 GCCACCAGCCCCTCCCCAGA CTCTCAGGTG GAGGCAACAG 40 50 base pairs nucleic acid singlelinear 15 GGGCTGTTGG CTCTGGTCTG CTCTGAAGGA AATTCCCTGG CCTTCCCTTG 50 15base pairs nucleic acid single linear 16 ACCAGAGCCA ACAGC 15 15 basepairs nucleic acid single linear 17 CCTGGCCTTC CCTTG 15 95 base pairsnucleic acid single linear 18 GGCCAGGCAT GGTAAGTCAT ACCTATAATCCCAGCACTGT GGGAGGCCAA GGAAGGGGGA 60 TCCCTTGAGC TCAAGAGTTT AAGACCGAGATCGAT 95 227 base pairs nucleic acid single linear cDNA 19 AAAGAGTTTAAGACCAGCTT GGGCAACACA GTCAGACTTC ATCTCTATAA ATAATTTAAA 60 AATTAGCCAAGCATGGTGGC GTGGTACCCT TGTGGGTTCC AGGCTTATTT GGGAGGTTGA 120 GGTAAAGGAATTCTCTTGGA CGCCCAGGTA GTCAAGGTTG CAGTGAGCCA TAATCAAACC 180 ACTGCACTCCAGCATGGCAA CAGAGCAAGA CCCCATCTCA AATATAT 227

1. A method of treating a disease condition in a patient selected fromthe group consisting of infectious diseases, genetic diseases,degenerative diseases, DNA damage, neoplasia, and skin diseases,comprising administering to the patient an effective amount of atherapeutic compound comprising a nucleic acid component of defibrotide,but not including defibrotide.
 2. A method of treating a diseasecondition in a patient selected from the group consisting of infectiousdiseases, genetic diseases, degenerative diseases, DNA damage,neoplasia, and skin diseases comprising the following steps: (a)determining the initial state of a set of disease markers associatedwith the disease condition, the disease markers being clinicallyobservable characteristics of a patient which deviate from the normalcondition due to the disease state and wherein each disease marker inthe set has a predetermined reference range which is indicative of thenormal condition, (b) administering to the patient a dose of atherapeutic compound comprising a nucleic acid component of defibrotide,but not including defibrotide, (c) screening a panel of secondmessengers and signal transducers and selecting a repair marker, theintensity of which increases following administration of the therapeuticcompound, where intensity is the extent to which the state of the repairmarker differs from its state in the normal condition, said repairmarker being the concentration of a compound which participates in acellular regulatory pathway which operates through protein kinase A,protein kinase C, or G-protein, (d) administering the therapeuticcompound at a dose level incrementally higher than the previous dose,(e) repeating step (d) each time the intensity of the repair markerincreases following an incrementally higher dose, (f) repeating steps(d) and (e) until the intensity of the repair marker in step (c) nolonger increases, (g) continuing administration of the therapeuticcompound at the highest dose level attained in step (f) until theintensity of the repair marker returns to the normal condition, and (h)administering the therapeutic compound at a dose level incrementallyhigher than the previous dose and repeating steps (c), (d), (e), (f) and(g) with one or more additional repair markers until all disease markersof said set of disease markers no longer deviate from the normalcondition.
 3. The method of claim 2 further comprising: (i) monitoringrepair markers selected in steps (c) and (h) for 3 weeks following thelast dose of the therapeutic compound given in step (h) and if theintensity of one or more repair markers deviate from the normalcondition, reinitiating therapy in step (g) at the highest dose levelachieved in step (h).
 4. A method of treating a disease condition in apatient selected from the group consisting of infectious diseases,genetic diseases, degenerative diseases, DNA damage, neoplasia, and skindiseases comprising the following steps: (a) determining the initialstate of a set of disease markers associated with the disease condition,the disease markers being clinically observable characteristics of apatient which deviate from the normal condition due to the disease stateand wherein each disease marker in the set has a predetermined referencerange which is indicative of the normal condition, (b) administering tothe patient a dose of a therapeutic compound comprising a nucleic acidcomponent of defibrotide, but not including defibrotide, (c) screening apanel of second messengers and signal transducers and selecting a repairmarker, the intensity of which increases following administration of thetherapeutic compound, where intensity is the extent to which the stateof the repair marker differs from its state in the normal condition, therepair marker being the concentration of a compound which participatesin a cellular regulatory pathway which operates through protein kinaseA, protein kinase C, or G-protein, (d) administering the therapeuticcompound at a dose level incrementally higher than the previous dose,(e) repeating step (d) each time the intensity of the repair markerincreases following an incrementally higher dose, (f) repeating steps(d) and (e) until the intensity of the repair marker in step (c) nolonger increases, (g) administering the therapeutic compound at the doselevel where the intensity of the repair marker no longer increases untilthe intensity of the repair marker returns to the normal condition, (h)administering the therapeutic compound at a dose level incrementallyhigher than the previous dose and repeating steps (c), (d), (e), (f) and(g) with one or more additional repair markers until all disease markersof said set of disease markers no longer deviate from the normalcondition, and (i) administering the therapeutic compound at a doselevel incrementally higher than the previous dose given in step (h) andrepeating steps (c), (d), (e), (f) and (g) until the intensity of auniversal marker returns to the normal condition, the universal markerbeing a constitutively expressed molecule which is transcriptionallyactivated by the therapeutic compound in all disease states.
 5. Themethod of claim 4 further comprising: (j) monitoring the universalmarker for 3 weeks following the last dose given in step (i) and if theintensity deviates from the normal condition, reinitiating therapy atstep (i) at the highest dose level achieved in step (i).
 6. A method oftreating a disease condition in a patient selected from the groupconsisting of infectious diseases, genetic diseases, degenerativediseases, DNA damage, neoplasia, and skin diseases comprising thefollowing steps: (a) determining the initial state of a set of diseasemarkers associated with the disease condition, the disease markers beingclinically observable characteristics of a patient which deviate fromthe normal condition due to the disease state and wherein each diseasemarker in the set has a predetermined reference range which isindicative of the normal condition, (b) administering to the patient adose of a therapeutic compound comprising a nucleic acid component ofdefibrotide, but not including defibrotide, wherein the dose of thetherapeutic compound is at a level which raises a universal marker to atleast five times its normal level, the universal marker being aconstitutively expressed molecule which is transcriptionally activatedby the therapeutic compound in all disease states, and (c) continuing toadminister the therapeutic compound at the dose level of step (b) untilthe universal marker returns to its normal level.
 7. The method of claim6 wherein the universal marker is vWAg.
 8. The method of claim 7 furthercomprising: (d) monitoring the universal marker for 3 weeks followingthe last dose given in step (c) and if the intensity deviates from thenormal condition, reinitiating therapy at step (c).
 9. The method ofclaim 2, wherein the disease condition is HIV infection, wherein HIV isnot expressed by said patient, and the concentration of at least oneimmunological molecule is elevated above the normal level comprising: a)administering to the patient an effective amount of a therapeuticcompound comprising a nucleic acid component of defibrotide, but notincluding defibrotide, wherein the effective amount is the amount whichcauses a universal marker to rise at least five times its normal level,the universal marker being the concentration of a constitutivelyexpressed molecule which is transcriptionally activated by thetherapeutic compound in all disease states, and b) continuing toadminister the effective amount of the therapeutic compound until theuniversal marker returns to its normal level.
 10. The method of claim 9wherein said immunological molecule is selected from the groupconsisting of CD4, CD25, IL1, IL-3, IL-4, IL-6, TNF and sIL2R.
 11. Themethod of claim 9 wherein the universal marker is the concentration ofvWAg.
 12. A method of treating a patient having a HIV associated diseasestate selected from the group consisting of tuberculosis, chronicwasting syndrome, and Herpesvirus infection comprising administering toan individual in need thereof an effective amount of a therapeuticcompound comprising a nucleic acid component of defibrotide, but notincluding defibrotide.
 13. A method of stimulating tissue repairassociated with HIV infection comprising: administering to a patient inneed thereof, an effective amount of a therapeutic compound comprising anucleic acid component of defibrotide, but not including defibrotide.14. The method of claim 2 wherein the disease condition is HIV infectionand wherein said disease marker is selected from the group consisting ofodynophagia, anthralgia, Herpes labialis, Herpes genitalis,cryptosporidium diarrhea, Karnofsky performance score, waste syndrome,oral and pharyngeal candidiasis, and tuberculosis.
 15. The method ofclaim 2 wherein said repair marker is selected from the group consistingof cAMP, cGMP, IL-1, IL-2, TNF-α, IL-6, cGMP/cAMP ratio, totallymphocyte count, T lymphocyte count, CD4 count, CD8 count, cAMPdependent protein kinase A enzyme, adenylate cyclase, G-protein,phosphoinositol, protein kinase C enzyme, inositol triphosphate,diacylglycerol, intracellular calcium level, intracellular calcium ionlevel, c-myc, ras, c-fos, c-jun, NK-kB, EIAI, AP-1, COUP, TCF-1α, TATA,TAT element, oxygen radical, CREB, CREM, Platelet Derived Growth Factor(PDGF), Colony Stimulating Factor (CSF), Epidermal Growth Factor (EGF),Insulin Growth Factor (IGF), cytosolic tyrosine kinase, src, SrcHomology 2 (SH2) domain, Src Homology 3 domain (SH3), serine/threoninekinase, Mitogen Activated Protein Kinase (MAP Kinase), Cytokine ReceptorSuperfamily, Signal Transducers and Activators of Transcription (STATs),JAJ1, JAK2, Tumor Necrosis Factor-Receptor 1 signal Transducer TRADD,chemokines of Rantes, and MIP-Alpha, and MIP-Beta.
 16. The method ofclaim 1 wherein the nucleic acid component of defibrotide is anoligonucleotide from about 6 nucleotides to less than 60 nucleotides inlength.
 17. The method of claim 1 wherein the nucleic acid component ofdefibrotide is selected from the group consisting of dCTP, dATP, dGTP,dTTP, dAMP, dGMP, dCDP, dADP, ATP, AMP, CTP, CMP, UTP, cyclic TMP,cyclic UMP, cyclic GMP, GGTTGGATTGGTTGG (SEQ ID NO:1), GGTTGGATCGGTTGG(SEQ ID NO:2), GGATGGATCGGTTGG (SEQ ID NO:3) and GGTGGTGGTTGTGGT (SEQ IDNO:4).
 18. The method of claim 1 wherein the nucleic acid component is avariant of an oligonucleotide selected from the group consisting ofGGTTGGATTGGTTGG, (SEQ ID NO:1) GGTTGGATCGGTTGG, (SEQ ID NO:2)GGATGGATCGGTTGG (SEQ ID NO:3) and GGTGGTGGTTGTGGT. (SEQ ID NO:4)


19. The method of claim 1 wherein the nucleic acid component is anoligonucleotide comprising the sequence of GGTGGTGGTTGTGGT (SEQ ID NO:4)and wherein said oligonucleotide is not a naturally existing nucleicacid component of defibrotide.
 20. The method of claim 18, wherein thevariant containing a sequence selected from the group consisting of HIVsequences, sequences encoding cellular regulatory factors andmitochondrial sequences.
 21. The method of claim 20, wherein the variantis selected from the group consisting ofGGGCTGTTGOCTCTGGTCTGCTCTGAAGGAAATTCCCTGGCCTTCCCTTG, (SEQ ID NO:15)ACCAGAGCCAACAGC, (SEQ ID NO:16) and CCTGGCCTTCCCTTG. (SEQ ID NO:17)


22. The method of claim 1 wherein the nucleic acid component is anoligonucleotide from about 25 nucleotides to about 30 nucleotides longand has a molecular weight of about 8171.58 Dalton.
 23. The method ofclaim 1 wherein the nucleic acid component is an oligonucleotide fromabout 25 nucleotides to about 30 nucleotides long and has a molecularweight of about 8433.75 Dalton.
 24. The method of claim 1 wherein thenucleic acid component of defibrotide is administered in combinationwith one or more sequence specific nucleic acid.
 25. The method of claim1 wherein the sequence specific nucleic acid is selected from the groupconsisting of an anti-protease sequence, a retroviral promoter sequence,a TAR sequence, a HIV mutant of TAR decoy RNA, a mutant TAR decoy RNA, anegative mutant of the viral REV transactivator, a synthetic promoterwith the consensus sequence for binding of the transcription factor aSp1 and the TATA box, a mutant of TATA box, a TAT mutant wherein themutations involving the seven cysteine residues, a sense, anti-sense,missense derivative of CIS acting negative elements (CRS) present in theintegrase gene and REV mutant, a transdominant suppressor of REV(mutations involving amino acid 78 and 79), a NEF-cDNA sequence and itsmutant with or without U3 region sequence of the 3′LTR, a POL reversetranscriptase gene mutant, a POL viral integrase gene and its mutant, aPOL viral protease gene mutant, a HIV-I LTR enhancer (−137 to −17)mutant, a HIV LTR promoter staring at −78, a HIV LTR sequence encoding aarginine fork from aa27 to aa38, a HIV-LTR sense sequence of thenegative regulatory element (−340 to −185), a HIV-1 LTR consensussequence for binding of transcription factors of AP1/COUP, NFAT-1, USF,TCF-α, NF-KB, TCF-1a, TBP, and an inhibitor of the consensus sequence, aLTR NFkB mutant (−104 to −80), a LTR Sp1 (GC box) binding site and TATAbox mutant, a LTR GAG gene sequence mutant, a LTR mutant (−454 to +180),a LTR genomic repeat at +80, a LTR region responsive for cellulartranscription factors between and to the left of U3 to −454 extending to−7, a 3′ LTR and its variant, a 5′ LTR and its variant, a LTR variant,an inhibitor of UBP-1 or LBP-1 binding sequence (−5 to +82), a ENV, GAG,POL gene sequences placed 3′ of the REV mutant codon, a short sequencemutant (15-60 mer) and a host DNA sequence of preferred targets forproviral integration.
 26. The method of claim 1 wherein said nucleicacid component of defibrotide is a nucleic acid derivative.
 27. Themethod of claim 1 wherein the dose of the therapeutic compound is fromabout 0.1 mg/kg patient body weight per day to about 1000 mg/kg patientbody weight per day.
 28. The method of claim 1 wherein the dose of thetherapeutic compound is from about 40 mg/kg patient body weight per dayto about 600 mg/kg patient body weight per day.
 29. The method of claim1 wherein the nucleic acid component of defibrotide is administered incombination with an amino acid selected from the group consisting ofthreonine, serine, tyrosine, and proline.
 30. The method of claim 1wherein the nucleic acid component of defibrotide is administered incombination with a N-containing ring compound selected from the groupconsisting of pyrimidine, purine, adenylic acid, and guanosine.
 31. Amethod of treating a disease condition in a patient selected from thegroup consisting of infectious diseases, genetic diseases, degenerativediseases, DNA damage, neoplasia, and skin diseases, comprisingadministering to the patient an effective amount of a therapeuticcompound comprising an oligonucleotide containing a homologous sequenceof HIV and a gene encoding a cellular regulatory factor.
 32. The methodof claim 31 wherein the cellular regulatory factor is selected from thegroup consisting of human TNF receptor, mouse TNF-receptor CCCR5, humanRIP protein kinase, IL-2 receptor, TNF receptor/cell death protein,IL-1a, TNF-α, c-myc, c-abl, c-fos, c-ras, dystrophin, surfaceglycoprotein proteins of L-CAM and cathedrin, and B-myb.
 33. The methodof claim 32 wherein the oligonucleotide is selected from the groupconsisting of CAGCTGCACCTGCCAAGC, (SEQ ID NO:5) ATAAAATATACCATATACA,(SEQ ID NO:6) TCATAAAATATACTATATTCA, (SEQ ID NO:7)ATATTAAAGAACGCTGTTTACAATACTTGG, (SEQ ID NO:8) ATGCAGTTGTGAAGAGAA, (SEQID NO:9) AATTAAGGCATAAGAAAACTAAGAAATATGCAC, (SEQ ID NO:10)TCTCTCCCTCAAGGACTCAGCTTTCTGAAG, (SEQ ID NO:11) CAATAATAAAAGGGGAAA, (SEQID NO:12) AGTGCAACCGGCAGGAGGTGA, (SEQ ID NO:13) andGCCACCAGCCCCTCCCCAGACTCTCAGGTGGAGGCAACAG. (SEQ ID NO:14)


34. The method of claim 31 wherein the oligonucleotide is administeredin combination with a homologous sequence of a gene encoding a cellularregulatory factor and GGTGGTGGTTGTTGGT (SEQ ID NO:4).
 35. The method ofclaim 34 wherein the cellular regulatory factor is selected from thegroup consisting of myc, TNF receptor, ras, abl, bcl, fos, IL-1, andmusnos.
 36. An oligonucleotide consisting of a sequence selected fromthe group consisting of CAGCTGCACCTGCCAAGC, (SEQ ID NO:5)ATAAAATATACCATATACA, (SEQ ID NO:6) TCATAAAATATACTATATTCA, (SEQ ID NO:7)ATATTAAAGAACGCTGTTTACAATACTTGG, (SEQ ID NO:8) ATGCAGTTGTGAAGAGAA, (SEQID NO:9) AATTAAGGCATAAGAAAACTAAGAAATATGCAC, (SEQ ID NO:10)TCTCTCCCTCAAGGACTCAGCTTTCTGAAG, (SEQ ID NO:11) CAATAATAAAAGGGGAAA, (SEQID NO:12) AGTGCAACCGGCAGGAGGTGA, (SEQ ID NO:13)GCCACCAGCCCCTCCCCAGACTCTCAGGTGGAGGCAACAG. (SEQ ID NO:14)GGGCTGTTGGCTCTGGTCTGCTCTGAAGGAAATTCCCTGGCCTTCCCTTG, (SEQ ID NO:15)ACCAGAGCCAACAGC, (SEQ ID NO:16) and CCTGGCCTTCCCTTG. (SEQ ID NO:17)


37. An oligonucleotide comprising the same sequence of anoligonucleotide of defibrotide obtainable via passing defibrotidethrough a C8 HPLC column and eluting with 0.1% TFA in water, wherein thelength of the oligonucleotide of defibrotide is from about 25 to about30 nucleotides and the molecular weight is 8171.58 Dalton, and whereinthe oligonucleotide is not a naturally existing nucleic acid componentof defibrotide.
 38. A vector comprising an origin of replication and asequence of the oligonucleotide in claim
 36. 39. The vector of claim 38further comprising a sequence encoding an origin of replication ofmitochondrion.
 40. The vector of claim 39 wherein the sequence encodingan origin of replication of mitochondrion is from a human.
 41. Thevector of claim 40 wherein the sequence is selected from the groupconsisting of 5′ end of mitochondrial 12S RNA containing sequences fromnucleotide 72 to 1025 and mitochondrial DNA containing sequences fromnucleotide 1 to
 72. 42. The vector of claim 40 further comprising apromoter sequence selected from the group consisting of TAR promoter,HIV LTR promoter, and promoter of DNA polymerase.
 43. The vector ofclaim 42 further comprising a sequence encoding DNA polymerase.
 44. Themethod of claim 1,2,4,6, 12 or 13 wherein the therapeutic compoundcomprising the vector of claim
 38. 45. The method of claim 44 whereinthe therapeutic compound is administered in combination with DNApolymerase, protease inhibitor, or reverse transcriptase.
 46. A methodof treating a patient having a resistance to a drug comprisingadministering an effective amount of a nucleic acid component ofdefibrotide in combination with the drug.
 47. The method of 46 whereinthe drug is protease inhibitor.
 48. The method of claim 1 wherein thedisease condition is HIV infection.
 49. An oligonucleotide comprisingthe same sequence of an oligonucleotide of defibrotide obtainable viapassing defibrotide through a C8 HPLC column and eluting with 0.1% TFAin water, wherein the length of the oligonucleotide of defibrotide isfrom about 25 to about 30 nucleotides and the molecular weight is8433.75 Dalton, and wherein the oligonucleotide is not a naturallyexisting nucleic acid component of defibrotide.
 50. A method of treatinga disease condition in a patient selected from the group consisting ofinfectious diseases, genetic diseases, degenerative diseases, DNAdamage, neoplasia, and skin diseases, comprising administering to thepatient an effective amount of defibrotide in combination with one ormore sequence specific nucleic acid.
 51. The method of claim 50 whereindefibrotide is administered in combination with one or more sequencespecific nucleic acid and one or more sequence specific peptide.
 52. Themethod of claim 51, wherein a nucleic acid component of defibrotide isadministered in combination with one or more sequence specific nucleicacid and one or more sequence specific peptide.